Acid addition salt of carbasugar amine derivative

ABSTRACT

An acid addition salt of a carba-sugar amine derivative represented by the following formula (1):  
                 
 
wherein R 1  and R 2  each independently represents a hydrogen atom, or an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group or an aralkyl group which may have one or at least two of the following substituent (I) or (II), or R 1  and R 2  are taken together to represent a substituent (III)  
                 
 
(R 8  to R 12  each independently represents an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group or an aralkyl group), with the proviso that both R 1  and R 2  are not a hydrogen atom at the same time; R 3 , R 4  and R 7  each independently represents a hydroxyl group or a hydroxyl group having a substituent; and R 5  and R 6  each independently represents a hydrogen atom, or a hydroxyl group or a hydroxyl group having a substituent, with the proviso that when one of R 5  and R 6  is a hydrogen atom, the other is a hydroxyl group or a hydroxyl group having a substituent.

TECHNICAL FIELD

The present invention relates to acid addition salts of novelcarba-sugar amine derivatives.

BACKGROUND OF THE INVENTION

G_(M1) gangliosidosis, ceramide lactoside lipidosis, Morquio B disease,Krabbe disease, Fabry disease, Gaucher disease, Tay-Sachs disease,Sandhoff disease, fucosidosis and the like are conventionally known asglycolipid metabolic disorders. These diseases are diseases which aregenerated as a result of causing mutation in various glycolytic enzymes.Among them, G_(M1) gangliosidosis, Morquio B disease, ceramide lactosidelipidosis and Krabbe disease are diseases which are generated whenβ-galactosidase loses its enzyme activity by undergoing mutation, andGaucher disease is a disease generated when β-glucosidase loses itsenzyme activity by undergoing mutation Carba-sugar amine derivative areknown as substances which have a possibility of becoming medicaments forthese diseases (International Publication WO 03/022797).

The carba-sugar amine derivatives described in International PublicationWO 03/022797 have a function to recover reduced or lost activity of amutated enzyme, but being considerably low in their solubility in water,it could not be said that they are sufficient for using as medicaments.

DISCLOSURE OF THE INVENTION

In order to solve the above-described problems, the present inventorshave conducted intensive studies and, as a result, discovered an acidaddition salt of a specified carba-sugar amine derivative having strongβ-galactosidase inhibitory activity or β-glucosidase inhibitoryactivity, and have found that this has excellent solubility in aqueoussolvents, thus accomplishing the invention.

That is, the present invention relates to the followings:

-   (1) An acid addition salt of a carba-sugar amine derivative    represented by the following formula (1):

wherein R¹ and R² each independently represents a hydrogen atom, or analkyl group, an alkenyl group, an alkynyl group, an acyl group, an arylgroup or an aralkyl group which may have one or at least two of thefollowing substituent (I) or (II), or R¹ and R² are taken together torepresent a substituent (III):

wherein R⁸ to R¹² each independently represents an alkyl group, analkenyl group, an alkynyl group, an acyl group, an aryl group or anaralkyl group;

with the proviso that both R¹ and R² are not a hydrogen atom at the sametime;

R³, R⁴ and R⁷ each independently represents a hydroxyl group or ahydroxyl group having a substituent; and

R⁵ and R⁶ each independently represents a hydrogen atom, a hydroxylgroup or a hydroxyl group having a substituent, with the proviso thatwhen one of R⁵ and R⁶ is a hydrogen atom, the other is a hydroxyl groupor a hydroxyl group having a substituent.

-   (2) An acid addition salt of a carba-sugar amine derivative    represented by the following formula (1)-A-2:

wherein R¹ and R² each represents as defined in the above-described (1).

-   (3) An acid addition salt of a carba-sugar amine derivative    represented by the following formula (1)-B-2;

wherein R¹ and R² each represents as defined in the above-described (1).

-   (4) The acid addition salt of a carba-sugar amine derivative    according to any one of (1) to (3), which is hydrochloride or    sulfate.-   (5) A medicament which comprises the acid addition salt of a    carba-sugar amine derivative according to any one of (1) to (4) as    an active ingredient.-   (6) The medicament according to (5), which is a preventive agent or    a therapeutic agent for glycolipid metabolic disorders.-   (7) A process for producing an acid addition salt of a carba-sugar    amine derivative, which comprises allowing a carba-sugar amine    derivative represented by the following formula (1) to contact with    an acid in an aqueous solvent to obtain the acid addition salt    thereof:

wherein R¹ and R² each independently represents a hydrogen atom, or analkyl group, an alkenyl group, an alkynyl group, an acyl group, an arylgroup or an aralkyl group which may have one or at least two of thefollowing substituent (I) or (II), or R¹ and R² are taken together torepresent a substituent (III):

wherein R⁸ to R¹² each independently represents an alkyl group, analkenyl group, an alkynyl group, an acyl group, an aryl group or anaralkyl group;

with the proviso that both R¹ and R² are not a hydrogen atom at the sametime;

R³, R⁴ and R⁷ each independently represents a hydroxyl group or ahydroxyl group having a substituent; and

R⁵ and R⁶ each independently represents a hydrogen atom, a hydroxylgroup or a hydroxyl group having a substituent, with the proviso thatwhen one of R⁵ and R⁶ is a hydrogen atom, the other is a hydroxyl groupor a hydroxyl group having a substituent.

The present invention is described below in more detail based on theembodiments of the present invention.

The present invention relates to an acid addition salt of a carba-sugaramine derivative represented by the above-described formula (1)(hereinafter referred to as the “substance of the invention”).

In this formula, R¹ and R² each independently represents a hydrogenatom, or a group generally used in the field of organic chemistry formodifying functional groups or protecting side chains, such as an alkylgroup, an aryl group or an aralkyl group, which may have one or at leasttwo of the following substituent (I) or (II), or R¹ and R² are takentogether to represent a substituent (III):

R⁸ to R¹² each independently represents an alkyl group, an alkenylgroup, an alkynyl group, an acyl group, an aryl group or an aralkylgroup, wherein both R¹ and R² are not a hydrogen atom at the same time.

The above-described alkyl group includes linear or branched alkyl groupshaving from 1 to 30, preferably from 2 to 23 carbon atoms. Particularly,when a medicament comprising the substance of the present invention asthe active ingredient (hereinafter referred to as the “medicament of theinvention”) is applied to a disease which is generated by an abnormalityof a sphingoglycolipid metabolic system, it is preferable that thestructure of the active ingredient can become a sphingoglycolipidanalogue. That is, as a sphingoglycolipid analogue (a substance whichhas a structure similar to that of sphingoglycolipid and performs anaction similar to or competitive with that of sphingoglycolipid) mainlyexisting in the living body, it is most desirable that R¹ in theabove-described formula has a linear alkyl group particularly havingfrom 8 to 23 carbon atoms.

Regarding the branched alkyl group among the above-described alkylgroups, a substituent may be introduced into the alkyl group, andexamples of the substituent include an alkoxy group, an aryloxy group,an amino group, a hydroxy group, a silyl group and the like. In general,the branched alkyl group may have a structure in which, for example, alinear alkyl group (it may also be a branched alkyl group) is furtherbound by a ether bond to a backbone in which a hydrogen atom bound to acarbon of an alkyl group such as an alkyl glycerol is substituted with ahydroxyl group (cf, the following formula (2)) (m in the followingstructural formula is each independently an integer of from 0 to 30) orthe like:

It is preferable that the above-described alkenyl group and alkynylgroup have from 1 to 30, preferably from 2 to 23, carbon atoms, and theymay have at least two carbon-to-carbon double bond or triple bond. Theabove-described acyl group may be any group, so long as it is a groupgenerally represented by —CO—R, and the number of carbon atoms as awhole acyl group is from 1 to 30, preferably from 2 to 23. In thisconnection, R in the above-described formula is any group selected fromthe above-described alkyl group, alkenyl group and alkynyl group and thearyl group and aralkyl group described below.

Also, the above-described aryl group includes an aryl group having from6 to 22, preferably from 6 to 14, carbon atoms, and examples includearomatic hydrocarbon residues such as a phenyl group or a naphthylgroup, or aromatic hydrocarbon residues in which a substituent such asan alkyl group or an acyl group is further substituted with a hydrogenatom of the aromatic ring (e.g., tolyl group and the like).

The above-described aralkyl group is a group having a general structureof Ar—(CH₂)_(n)— in which a hydrogen atom of an alkyl group issubstituted with an aryl group, and the above-described n is preferablyfrom 1 to 30, more preferably from 2 to 23. Examples of the aralkylgroup include a benzyl group, a phenetyl group, an α-methylbenzyl groupand the like.

Examples of the above-described alkyl group, alkenyl group, alkynylgroup, acyl group, aryl group or aralkyl group which may have (I) or(II) having the above-described substituent include compounds of thefollowing (A) to (D), and examples of the compound having thesubstituent (III) in which R¹ and R² are combined include the followingcompounds.

(In the formulae, R¹³ to R¹⁵ each represents a hydrogen atom, or analkyl group, an alkenyl group, an alkynyl group, an acyl group, an arylgroup or an aralkyl group having from 0 to 30 carbon atoms.)

When R³, R⁴ and R⁷ each is a hydroxyl group or a hydroxyl group having asubstituent, a hydroxyl group is particularly preferred.

However, when one of R⁵ and R⁶ is a hydroxyl group or a hydroxyl grouphaving a substituent, the other is a hydrogen atom. Each of them is nota hydroxyl group nor hydroxyl group having a substituent at the sametime. Also, when R⁵ and R⁶ each independently is a hydrogen atom, ahydroxyl group or a hydroxyl group having a substituent, hydroxyl groupis particularly preferred.

In this connection, examples of the substituent of the hydroxyl groupinclude single substituents such as aralkyl groups (benzyl group,phenetyl group, α-methylbenzyl group and the like), silyl groups(trimethylsilyl group, triethylsilyl group, triisopropylsilyl (TIPS)group, t-butyldiphenylsilyl (TBDPS) group, t-butyldimethylsilyl (TBDMS)group and the like), alkanoyl groups (acetyl group, butyryl group andthe like), aroyl groups (benzoyl group, toluoyl group, naphthoyl groupand the like), alkoxyalkyl groups (methoxymethyl (MOM) group and thelike), and aralkyloxyalkyl groups (benzyloxymethyl (BOM) group and thelike), and a group in which two of R³ to R⁷ together form a substituentsuch as alkylidene groups (methylidene group, ethylidene group and thelike), an isopropylidene group and aralkylidene groups (benzylidenegroup and the like). Among these, MOM is particularly preferred from theviewpoint of its stability and easy handling and leaving.

When each of R³, R⁴, R⁶ and R⁷ is a hydroxyl group and R⁵ is a hydrogenatom in a carba-sugar amine derivative represented by theabove-described formula (I), it can be represented by the followingformula (I)-A-2 (corresponding to Compounds 25-1 to 25-7 in FIG. 1).

Also, when each of R³, R⁴, R⁵ and R⁷ is a hydroxyl group and R⁶ is ahydrogen atom in a carba-sugar amine derivative represented by theabove-described formula (I), it can be represented by the followingformula (I)-B-2 (corresponding to Compounds 30-1 to 30-7 in FIG. 1).

The substance of the invention is an acid addition salt of the compoundrepresented by the above-described formula (I), and examples of such anacid addition salt include salts with acids such as inorganic acids(sulfuric acid, nitric acid, phosphoric acid, hydrohalogenic acids(hydrochloric acid, hypochlorous acid, hydrobromic acid and the like)and organic acids (acetic acid, propanoic acid, hydroxyacetic acid,2-hydroxypropanoic acid, 2-oxopropanoic acid, ethanedioic acid,propanedioic acid, butanedioic acid, (Z)-butanedioic acid,(E)-2-butanedioic acid, 2-hydroxybutanedioic acid,2,3-dihydroxybutanedioic acid, 2-hydroxy-1,2,3-propanetricarbonic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,4-methylbenzenesulfonic acid, toluenesulfonic acid, cyclohexanesulfamicacid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid, oxalic acid,maleic acid, malonic acid, succinic acid, fumaric acid, mandelic acid,tartaric acid, malic acid, ascorbic acid, citric acid, lactic acid,butyric acid, salicylic acid, nicotinic acid and the like). Among these,inorganic acid salts are preferred, and hydrochloride or sulfate isparticularly preferred.

In this connection, since the substance of the invention is an acidaddition salt of the compound represented by formula (1) which is a kindof pseudo-sugar, carbon numbers of the compound in this description aredescribed by the method shown in the following formula (3) in accordancewith the case of hexose;

In addition, regarding the substituted amino group represented by NR¹R²in formula (1), when the substance of the invention as a pseudo-sugar isassumed to be a hexose (when, in the above-described formula (3), thecarbon shown by 5a linked to the 1-position and 5-position is assumed tobe the oxygen of the 6-membered ring), a case in which the substitutedamino group (NR¹R²) is on the upper side against the 6-membered ring isdefined as β type, and is on the opposite side is defined as α type, andit is preferable that the substance of the invention is β type.

Since the substance of the invention is a salt of a substance havinghigh inhibitory activity upon a β-galactosidase or β-glucosidase derivedfrom a mammal, particularly human, it is possible to use the substanceas a reagent which inhibits these enzymes in vitro or in vivo (cells,tissues and the like), a medicament based on such an enzyme inhibitoryaction or a medicament for treating (treating or preventing) glycolipidmetabolic disorders. Since the substance of the invention hasconsiderably higher solubility in water-soluble solvents, such as water,than that of the conventionally known free type substances, it isconsidered that its blood concentration can be increased and reductionof its dose can be attained when used particularly as a medicament, andpreparation of various medicaments becomes easy. In addition, thesubstance of the invention can also be used in the studies on pathologyof diseases induced by the mutation of β-galactosidase or β-glucosidase.

In this connection, the inhibitory activity upon β-galactosidase can becalculated by adding the substance of the invention to a solutioncontaining β-galactosidase and its substrate, or to a solutioncontaining β-glucosidase and its substrate in the case of the inhibitoryactivity upon β-glucosidase, and comparing the respective enzymeactivities with the case of not adding the substance of the invention.

It is preferable that the substance of the invention is a salt of asubstance having a 50% inhibition concentration (IC₅₀) of less than 1μmol/l against the activity of mammalian β-galactosidase orβ-glucosidase, and it is particularly preferable that the IC₅₀ is lessthan 0.5 μmol/l.

In addition, the substance of the invention can be synthesized byallowing a compound represented by the formula (1) (e.g., formula(1)-A-2 or (1)-B-2) obtained by the method described in InternationalPublication WO 03/022797 or the synthesis route of FIG. 1 to react with,for example, 1 to 6 mol/l of an acid exemplified above (e.g.,hydrochloric acid, acetic acid and the like) in an aqueous solvent.After the above-described reaction, the substance of interest can beobtained by a method in which this is azeotroped preferably with organicsolvents (e.g., ethanol and toluene), and the residue is dissolved inwater and freeze-dried.

The medicament of the invention may contain other components, so long asit contains an effective amount of the substance of the invention Themedicament of the invention can be produced, for example, by combiningthe substance of the invention with a pharmaceutically acceptablecarrier. Although the carrier is not particularly limited, examplesinclude carriers which are generally used in medicaments such asexcipients, binders, disintegrating agents, lubricants, stabilizingagents, correctives, diluents, surfactants, and solvents for injection.

Dosage forms of the medicament of the invention are not particularlylimited and can be optionally selected in response to the therapeuticobjects, and specific examples include tablets, pills, suspensions,emulsions, capsules, solutions, syrups, suppositories, injections,granules, powders, liposome forming agents, inhalation powders and thelike.

The medicament of the invention can be administered orally orparenterally to mammals including human. The administration period isnot particularly limited, and it is possible to select theadministration period optionally in response to the therapeutic methodof diseases to be treated. In addition, it is preferable to decide theadministration form in response to the preparation forms, age, sex andother conditions of each patient, degree of symptoms of each patient andthe like. Dose of the medicament of the invention is not particularlylimited and should be individually set depending on the kind andspecific activity of the active ingredient, the kind, symptoms and thelike of the animal to be administered, and the kind of living bodytissue to be administered and its conditions and the like, but ingeneral, approximately from 0.1 μg to 1,000 mg can be administered perday as an acid addition salt of the compound represented by the formula(1).

Concentration of the active ingredient in the medicament of theinvention is optionally selected in response to the usage, the age andsex of each patient, degree of the disease, other conditions and thelike. In general, it is preferable that the concentration of themedicament of the invention is set to a range of from 0.001 to 5% (w/v)as the active ingredient. For example, when the medicament of theinvention is used as solutions for oral administration, it is preferably0.01% (w/v) or more, most preferably from 0.03% to 0.2% (w/v). Also,when used as injections for intramuscular injection or intravenousinjection, it is preferably 0.01% (w/v) or more, most preferably 0.03%(w/v) or more.

Since the substance of the invention has a specific and stronginhibitory activity for normal β-galactosidase or β-glucosidase derivedfrom a mammal and also has a function to restore activities of theseenzymes reduced or lost in the living body, it can be used for theexcellent treatment or prevention of glycolipid metabolic disordersbased on the mutation of the β-galactosidase or β-glucosidase gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a scheme for synthesizing the substance of theinvention. In the drawing, MOM represents a methoxymethyl group, and BOCrepresents a t-butoxycarbonyl group, Ac represents a acetyl group, Phrepresents a phenyl group, and MS represents methanesulfonic acid.

FIG. 2 is a graph showing neutral β-galactosidase inhibitory activity ofSubstances A-2 to 7 of the invention.

FIG. 3 is a graph showing neutral β-galactosidase inhibitory activity ofSubstances B-2 to 7 of the invention.

FIG. 4 is a graph showing acidic β-galactosidase inhibitory activity ofSubstances A-2 to 7 of the invention.

FIG. 5 is a graph showing neutral β-glucosidase inhibitory activity ofSubstances A-2 to 7 of the invention.

FIG. 6 is a graph showing neutral β-glucosidase inhibitory activity ofSubstances B-2 to 7 of the invention.

FIG. 7 is a graph showing ¹H-NMR charts of 25-2 and Substance A-2 of theinvention.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention is specifically described below based on examples.

<1> Synthesis of Compounds

(1) Synthesis of Compound 1

A starting material (methyl-α-D-glucopyranoside, manufactured by Sigma)(10 g, 51.493 mmol) was dissolved in dimethylformamide (150 ml), andimidazole (3.856 g, 56.642 mmol) was added thereto. Then, the mixturewas cooled to 0° C., and t-butyldimethylsilyl chloride (8.536 g, 56.642mmol) was added thereto, followed by stirring for 1.5 hours in argonatmosphere. After completion of the reaction, the solvent was evaporatedto obtain Compound 1 quantitatively.

TLC: Rf=0.46 (chloroform:methanol=4:1)

¹H-NMR (500 MHz; CDCl₃)

δ=3.778 (dd, 1H), 3.730 (dd, 1R), 3.647 (t, 1H), 3.512-3.481 (m, 1H),3.438-3.402 (m, 2H), 3.324 (s, 3H)

(2) Synthesis of Compound 2

Compound 1 (51.493 mmol) was dissolved in 1,2-dichloroethane (50 ml),and N-diisopropylethylamine (53.824 ml 308.994 mmol) was added thereto.Then, chloromethyl methyl ether (14.08 ml, 185.396 mmol) was addeddropwise thereto at 60° C. in argon atmosphere, followed by stirring for1.5 hours. After completion of the reaction, the solvent was evaporated,and the residue was diluted with ethyl acetate and washed with water andsaturated brine in this order. The solvent was evaporated, and theresidue was purified by silica gel column chromatography (toluene:ethylacetate=10:0-7:3) to obtain 19.971 g (88%) of Compound 2.

TLC: Rf=0.43 (toluene:ethyl acetate=1:1)

δ=4.822-4.682 (m, 7H), 3.882 (t, 1H), 3.854 (dd, 1H), 3.751 (dd, 1H),3.582-3.549 (m, 1H), 3.480-3.433 (m, 2H), 3.396 (s, 3H), 3.391 (s, 3H),3.380 (s, 3H), 3.376 (s, 3H)

(3) Synthesis of Compound 3

Compound 2 (19.971 g, 45.327 mmol) was dissolved in tetrahydrofuran(THF) (30 ml), and 1 M tetrabutylammonium fluoride/tetrahydrofuran(58.925 ml, 58.925 mmol) was added dropwise thereto, followed bystirring at room temperature for 40 minutes in argon atmosphere. Aftercompletion of the reaction, the solvent was evaporated, and the residuewas diluted with ethyl acetate and washed with water and saturated brinein this order. The solvent was evaporated, and the residue was purifiedby silica gel column chromatography (toluene:ethyl acetate=1:1-3:7) toobtain 13.298 g (90%) of Compound 3.

TLC: Rf=0.45 (toluene:ethyl acetate=2:1)

¹H-NMR (500 MHz, CDCl₃)

δ=4.930 (d, 1H, J_(gem)=6.4, CH₃OCH ₂—), 4.843 (d, 1H, J_(gem)=6.4,CH₃OCH ₂—), 4.833 (d, 1H, J₁₋₂=3.4, H-1), 4.782 (d, 1H, J_(gem)=6.4,CH₃OCH ₂—), 4.774 (d, 1H, J_(gem)=6.8, CH₃OCH ₂—), 4.714 (d, 1H,J_(gem)=6.8, CH₃OCH ₂—), 4.706 (d, 1H, J_(gem)=6.4, CH₃OCH ₂—), 3.942(t, 1H, J=9.3, H-4 or 3), 3.897 (ddd, 1H, J_(6B-5)=3.5, J_(6B-OH)=6.1,J_(gem)=12.6, H-6B), 3.789 (ddd, 1H, J_(6A-5)=2.2, J_(6A-OH)=8.3,J_(gem)=12.7, H-6A), 3.640 (ddd, 1H, J_(5-6A)=2.2, J_(5-6B)=3.3,J₅₋₄=10.1, H-5), 3.581 (dd, 1H, J=8.9, J=9.9, H-3 or 4), 3.524 (dd, 1H,J₂₋₁=3.8, J₂₋₃=9.6, H-2), 3.446, 3.415, 3.412, 3.400 (s, 3H, CH₃OCH₂—)×4, 2.646 (dd, 1H, J_(OH-6A)=8.2, J_(OH-6A)=6.2, OH)

(4) Synthesis of Compound 4

Compound 3 (13.298 g, 40.746 mmol) was dissolved in pyridine (45 ml),and triphenylphosphine (17.101 g, 65.198 mmol) was added thereto. Then,N-iodosuccinimide (14.608 ml, 65.198 mmol) was gradually added thereto,followed by stirring at room temperature for 4 hours in argonatmosphere. After completion of the reaction, the reaction was stoppedby adding methanol (30 ml), and the solvent was evaporated. The residuewas diluted with ethyl acetate and washed with saturated sodiumbicarbonate water and saturated brine in this order. The solvent wasevaporated, and the residue was purified by silica gel columnchromatography (toluene:ethyl acetate=7:3) to obtain 14.650 g (82%) ofCompound 4.

TLC: Rf=0.37 (toluene:ethyl acetate=3:1)

¹H-NMR (500 MHz, CDCl₃)

δ=4.922 (d, 1H, J_(gem)=6.6, CH₃OCH ₂—), 4.842 (d, 1H, J₁₋₂=3.7, H-1),4.824 (d, 1H, J_(gem)=6.1, CH₃OCH ₂—), 4.776 (d, 1H, J_(gem)=6.8, CH₃OCH₂—), 4.775 (d, 1H, J_(gem)=5.9, CH₃OCH ₂—), 4.742 (d, 1H, J_(gem)=6.6,CH₃OCH ₂—), 4.710 (d, 1H, J_(gem)=6.8, CH₃OCH ₂—), 3.930 (dd, 1H, J=9.0,J=9.8, H-3), 3.621 (dd, 1H, J_(6B-5)=2.6, J_(gem)=10.6, H-6B), 3.516(dd, 1H, J₂₋₁=3.5, J₂₋₃=9.9, H-2), 3.506 (m, 1H, H-5), 3.485, 3.428,3.407, 3.398 (s, 3H, CH ₃OCH₂—)×4, 3.311-3.355 (dd×2, 2H, H-4, H-6A)

(5) Synthesis of Compound 5

Compound 4 (3.998 g, 9.165 mmol) was dissolved in toluene (12 ml), andDBU (6.853 ml, 45.825 mmol) was added thereto, followed by stirring at50° C. for 17 hours in argon atmosphere. After completion of thereaction, the solvent was evaporated, and the residue was purified bysilica gel column chromatography (toluene; ethyl acetate=7.3) to obtain1.638 g (58%) of Compound 5.

TLC: Rf=0.54 (toluene:methanol=20:1)

¹H-NMR (500 MHz, CDCl₃)

δ=4.885 (d, 1H, J₁₋₂=3.4, H-1), 4.840 (m, 5H), 4.780 (d, 1H, J=6.6),4.734 (d×2, 2H), 4.030 (dt, 1H, J=2.0, J=9.3), 3.930 (t, 1H, J=9.40),3.649 (dd, 1H, J=3.4, 9.6, H-2), 3.469, 3.466, 3.427, 3.413 (s, 3H, CH₃O—)×4

(6) Synthesis of Compound 7

Compound 5 (180.8 mg, 0.5864 mmol) was dissolved in a mixed solution ofdioxane (6 ml) and water (4 ml), and palladium chloride (13.8 mg,0.07782 mmol) was added thereto, followed by stirring at 60° C. for 4hours. After completion of the reaction, the reaction solution wasconcentrated, diluted with 50 ml of saturated brine and extracted 5times with 50 ml of ethyl acetate. The resulting organic layer was driedover magnesium sulfate, and the solvent was evaporated under reducedpressure to obtain Compound 6 (141 mg). Compound 6 was dissolved inpyridine (3 ml), and acetic anhydride (3 ml) was added thereto, followedby stirring at 60° C. for 3.5 hours. After completion of the reaction,the solvent was evaporated, and the residue was purified by silica gelcolumn chromatography (toluene:ethyl acetate=4:1, 0.2% triethylamine) toobtain 127.4 mg (78.6%) of Compound 7.

TLC: Rf=0.42 (toluene:ethyl acetate=2:1)

¹H-NMR (500 MHz, CDCl₃)

δ=6.880 (dd, 1H, J=2.1, J=10.4), 6.055 (dd, 1H, J=2.4, J=10.5), 4.942(d, 1H, J=6.8, CH₃OCH ₂—), 4.929 (d, 1H, J=6.8, CH₃OCH ₂—), 4.903 (d,1H, J=6.3, CH₃OCH ₂—), 4.854 (d, 1H, J=6.8, CH₃OCH ₂—), 4.838 (d, 1H,J=6.6, CH₃OCH ₂—), 4.816 (d, 1H, J=6.8, CH₃OCH ₂—), 4.457 (dt, 1H,J=2.2, J=8.3), 4.229 (d, 1H, J=11.0), 3.993 (dd, 1H, J=8.3, J=11.0),3.489, 3.469, 3.443 (s, 3H, CH ₃OCH₂—)×3

(7) Synthesis of Compound 8

Compound 7 (127.4 mg, 0.4132 mmol) was dissolved in anhydroustetrahydrofuran (10 ml), and 0.5 M Tebbe Reagent/toluene (1.65 ml, 0.825mmol) was added thereto, followed by stirring for 1.5 hours underice-cooling. The reaction solution was diluted with ethyl acetate andwashed with saturated sodium bicarbonate water and saturated brine inthis order. The solvent was evaporated under reduced pressure, and theresulting residue was purified by silica gel column chromatography(toluene:ethyl acetate=6:1, 0.2% triethylamine) to obtain 54.5 mg ofCompound 8 (37.5% from Compound 5 by 3 steps).

TLC: Rf=0.53 (toluene:ethyl acetate=2:1)

¹H-NMR (500 MHz, CDCl₃)

δ=6.186 (dd, 1H, J=1.8, J=10.1), 5.723 (br.d, 1H, J=10.0), 5.290 (br.s,1H), 5.123 (br.s, 1H), 4.919 (d, 1H, J=6.6), 4.847 (m, 3H), 4.795 (d,1H, J=6.6), 4.769 (d, 1H, J=6.8), 4.263 (m, 2H), 3.782 (dd, 1H, J=7.0,J=9.4), 3.464, 3.435, 3.429 (s, 3H, CH ₃OCH₂—)×3

(8) Synthesis of Compound 10

Compound 8 (38.4 mg, 0.140 mmol) was dissolved in methanol (3 ml), andtrifluoroacetic acid (1.5 ml) was added thereto, followed by stirringunder heating at 70° C. for 5 hours. Trifluoroacetic acid (0.5 ml×3) wasfurther added thereto, followed by stirring for 4.5 hours. Aftercompletion of the reaction, the solvent was evaporated under reducedpressure to obtain Compound 9. Then, the resulting Compound 9 wasdissolved in pyridine (1.5 ml), and acetic anhydride (1.5 ml) was addedthereto, followed by stirring at room temperature for 1.5 hours. Aftercompletion of the reaction, the solvent was evaporated under reducedpressure, and the resulting residue was purified by silica gel columnchromatography (toluene:ethyl acetate=5:1) to obtain 29.4 mg (75%) ofCompound 10.

TLC: Rf=0.50 (toluene:ethyl acetate=3:1)

¹H-NMR (500 MHz, CDCl₃)

δ=6.280 (m, 1H), 5.703 (m, 1H), 5.628 (m, 2H), 5.293 (dd, 1H, J=7.8,J=10.5), 5.187 (m, 1H), 5.055 (m, 1H), 2.141, 2.068, 2.052 (s, 3H, CH₃C═O)×3

(9) Synthesis of Compound 11

In argon atmosphere, a carbon tetrachloride solution (1,670 ml) ofCompound 10 (20.11 g, 75.0 mmol) was stirred, and bromine (0.3128mmol/ml carbon tetrachloride solution, 237.3 ml, 74.3 mmol) was addeddropwise thereto over 6 hours. After further stirring 30 minutes, thereaction solution was diluted with carbon tetrachloride and washed withsaturated sodium bicarbonate water and water in this order. After dryingthe resulting organic layer over magnesium sulfate, the solvent wasevaporated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography (toluene ethylacetate=20:1) to obtain Compound 11 (30.11 g, 94%).

TLC: Rf=0.47, 0.52 (toluene:ethyl acetate=4:1)

Compound 11α

¹H-NMR (500 MHz, CDCl₃)

δ=6.196 (d, 1H, J=5.6), 6.016 (d, 1H, J=7.8), 5.655 (dd, 1H, J=7.8,J=10.7), 4.948 (m, 2H), 4.001 (d, 1H, J=10.7), 3.855 (d, 1H, J=10.7),2.131, 2.110, 2.065 (s, 3H, CH ₃C═O)×3

Compound 11β

¹H-NMR (500 MHz, CDCl₃)

δ=6.125 (br.s, 1H), 5.962 (m, 1H), 5.532 (dd, 1H, J=8.3, J=10.5), 5.228(dd, 1H, J=7.8, J=10.5), 4.671 (m, 1H), 4.016 (m, 1H), 3.834 (dd, 1H,J=0.5, J=10.7), 2.101, 2.088, 2.031 (s, 3CH ₃C═O)×3

(10) Synthesis of Compound 13

Potassium acetate (3.97 g, 40.5 mmol) was added to a dimethylformamidesolution (DMF) (2,610 ml) of Compound 11 (19.85 g, 46.4 mmol), followedby stirring for 2 hours, potassium acetate (579 mg, 5.90 mmol) wasfurther added thereto, followed by stirring for 2 hours, and sodiumazide (6.05 g, 93.1 mmol) was further added thereto, followed bystirring for 4 hours. The solvent of the reaction solution wasevaporated under reduced pressure, and the resulting residue was dilutedwith ethyl acetate and washed with water and saturated brine in thisorder. After drying the resulting organic layer with magnesium sulfate,the solvent was evaporated under reduced pressure, and the residue waspurified by silica gel column chromatography (toluene:ethylacetate=10:1) to obtain Compound 13 (8.74 g, 48%).

Compound 13αβ

TLC; Rf=0.23, 0.30 (toluene:ethyl acetate=5:1)

¹H-NMR (500 MHz, CDCl₃)

δ=5.929 (m, 1H, β), 5.771 (m, 2H, α), 5.659 (m, 1H, β), 5.499 (dd, 1H,J=7.2, J=10.6, β), 5.292 (m, 2H, α), 5.177 (dd, 1H, J=4.4, J=10.5, β),4.693 (m, 1H, α, 1H, β), 4.410 (m, 1H, α, 2H, β), 4.244 (m, 1H, α),2.105, 2.071, 2.046, 2.028 (s, 3H, CH ₃C═O, α)×4, 2.128, 2.064, 2.046(s, 3H, CH ₃C═O, β)×4

(11) Synthesis of Compound 14

In argon atmosphere, sodium methoxide (1.26 g, 23.3 mmol) was added to amethanol solution (1,000 ml) of Compound 13 (85.83 g, 232.4 mmol),followed by stirring at room temperature for 90 minutes. The reactionsolution was neutralized with Amberlist 15 DRY (trade name, manufacturedby Organo Corporation) and filtered through Celite. The filtrate wascombined with the resin after washing (tetrahydrofuran:MeOH=1:1), thesolvent was evaporated under reduced pressure, and the resulting residuewas purified by silica gel column chromatography(chloroform:methanol=5:1) to obtain Compound 14αβ (37.48 g, 93%).

TLC: Rf=0.24 (chloroform:methanol=5:1)

¹H-NMR (500 MHz, CD₃OD)

δ=5.771 (m, 0.4H), 5.526 (m, 1H), 4.154 (m, 2.8H), 4.093 (m, 1H), 3.970(m, 1.4H), 3.680 (m, 0.4H), 3.464 (m, 2H), 3.306 (m, 1H)

(12) Synthesis of Compound 15

In argon atmosphere, benzaldehyde dimethyl acetal (36.3 ml, 0.242 mmol)and p-toluenesulfonic acid (3.54 g, 18.6 mmol) were added to adimethylformamide solution (700 ml) of Compound 14 (37.48 g, 186 mmol),followed by stirring at room temperature for 1.5 hours and then at 45°C. for 3.5 hours. p-Toluenesulfonic acid (1.06 g, 5.57 mmol) was furtheradded thereto, followed by stirring for 3.5 hours. About 7 ml of thesolvent in the reaction solution was evaporated under reduced pressure,and the residue was stirred at 45° C. for 1 hour. The reaction solutionwas ice-cooled and stirred by adding triethyl amine (34.0 ml, 0.244mmol), and the solvent was evaporated under reduced pressure. Theresulting residue was purified by silica gel column chromatography(toluene:ethyl acetate=2:1, 0.5% triethylamine) and furtherrecrystallized from dichloromethane and diisopropyl ether to obtainCompound 15 (26.01 g, 72%).

TLC: Rf=0.44 (toluene:ethyl acetate=1:1)

¹H-NMR (500 MHz, CDCl₃)

δ=7.483 (m, 2H, Ph), 7.382 (m, 3H, Ph), 5.663 (s, 1H, CH-Ph), 5.471(br.s, 1H, H-5a), 4.495 (m, 2H, H-6A, H-6B), 4.451 (m, 1H, H-4), 4.184(m, 1H, H-1), 3.894 (dq, 1H, J=2.9, J=10.5, J=2.7, J=10.5, H-3), 3.730(dq, 1H, J=2.2, J=10.5, J=2.2, J=10.5, H-2), 2.887 (d, 1H, J=2.2, OH),2.850 (d, 1H, J=2.9, OH)

(13) Synthesis of Compound 16

In argon atmosphere, N-ethyldiisopropylamine (173 ml, 993 mmol) wasadded to a dichloroethane solution (450 ml) of Compound 15 (14.36 g,49.6 mmol), and chloromethyl methyl ether (37.7 ml, 496 mmol) was addeddropwise thereto, followed by stirring at room temperature for 15minutes and then at 60° C. for 6.5 hours. The reaction solution wasice-cooled, triethylamine (138 ml, 993 mmol) was added thereto, andethanol (77.7 ml, 1.34 mol) was added dropwise thereto, followed bystirring at room temperature for 35 minutes. The solvent in the reactionsolution was evaporated under reduced pressure, the resulting residuewas dissolved in ethyl acetate and washed with saturated sodiumbicarbonate water and saturated brine in this order, the resultingorganic layer was dried over magnesium sulfate, and the solvent wasevaporated under reduced pressure. The resulting residue was purified bysilica gel column chromatography (toluene:ethyl acetate=20:1, 0.5%triethylamine) to obtain

Compound 16 (18.18 g, 97%).

TLC: Rf=0.52 (toluene:ethyl acetate=4:1)

¹H-NMR (500 MHz, CDCl₃)

δ=7.469 (m, 2H, Ph), 7.355 (m, 3H, Ph), 5.650 (s, 1H, CH-Ph), 5.503(br.s, 1H, H-5a), 4.989 (d, 1H, J=6.6, CH₃OCH ₂—), 4.892 (d, 1H, J=6.4,CH₃OCH ₂—), 4.838 (d, 1H, J=6.4, CH₃OCH ₂—), 4.817 (d, 1H, J=6.4, CH₃OCH₂—), 4.528 (m, 1H), 4.472 (m, 2H), 4.079 (m, 1H), 3.980 (dd, 1H, J=7.7,J=10.6), 3.674 (dd, 1H, J=8.7, J=10.6), 3.508, 3.363 (s, 3H, CH₃OCH₂—)×2

(14) Synthesis of Compound 17

p-Toluene sulfonic acid (3.53 g, 18.6 mmol) was added to a methanolsolution (210 ml) of Compound 16 (14.0 g, 37.1 mmol), followed bystirring at room temperature for 20 minutes. The reaction solution wasice-cooled, triethylamine (25.8 ml, 185 mmol) was added thereto, and thesolvent was evaporated under reduced pressure. The resulting residue waspurified by silica gel column chromatography (chloroform:methanol=40:1,0.5% triethylamine) to obtain Compound 17 (8.67 g, 81%).

TLC; Rf=0.39 (chloroform:methanol=20:1)

¹H-NMR (500 MHz, CDCl₃)

δ=5.598 (br.s, 1H, H-5a), 4.905 (d, 1H, J=6.6, CH₃OCH ₂—), 4.826 (d, 1H,J=6.8, CH₃OCH ₂—), 4.781 (d, 1H, J=6.8, CH₃OCH ₂—), 4.767 (d, 1H, J=6.8,CH₃OCH ₂—), 4.569 (d, 1H, J=2.4), 4.294 (m, 1H), 4.259 (br.d, 1H,J=3.7), 4.184 (br.dd, 1H, J=7.2, J=13.1), 4.021 (m, 1H), 3.686 (dd, 1H,J=8.3, J=10.0), 3.531 (dd, 1H, J=7.3, J=10.0), 3.492, 3.480 (s, 3H, CH₃OCH₂—)×2, 2.572 (br.dd, 1H, J=4.6, J=7.8)

(15) Synthesis of Compound 19

Triethylamine (120 ml, 861.0 mmol) and methanesulfonic acid chloride (67ml, 865.7 mmol) were added to a dichloromethane solution (600 ml) ofCompound 17 (12.49 g, 43.2 mmol), followed by stirring at 0° C. for 45minutes. After completion of the reaction, triethylamine (150 ml) andmethanol (300 ml) were added thereto, and the solvent was evaporatedunder reduced pressure. The residue was dissolved in ethyl acetate andwashed with saturated sodium bicarbonate water and saturated brine inthis order, the resulting organic layer was dried over magnesiumsulfate, and the solvent was evaporated under reduced pressure to obtainCompound 18.

Then, Compound 18 was dissolved in toluene (600 ml), and cesium acetate(24.92 g, 129.8 mmol) and 18-6 crown ether (68.50 g, 259.2 mmol) wereadded thereto, followed by stirring at 90° C. for 80 minutes. Thereaction solution was diluted with ethyl acetate and washed withsaturated sodium bicarbonate water and saturated brine in this order,the resulting organic layer was dried over magnesium sulfate, and thesolvent was evaporated under reduced pressure. The resulting residue waspurified by silica gel column chromatography (toluene:ethyl acetate=4:1,0.2% triethylamine) to obtain Compound 19 (12.01 g, 74.5%).

TLC: Rf=0.41 (toluene:ethyl acetate=2:1)

¹H-NMR (500 MHz, CDCl₃)

δ=5.845 (m, 1H), 5.671 (d, 1H, J=3.7), 4.948 (d, 1H, J=6.6, CH₃OCH ₂—),4.770 (d, 1H, J=6.6, CH₃OCH₂—), 4.744 (d, 1H, J=7.1, CH₃OCH ₂—), 4.637(d, 1H, J=6.8, CH₃OCH ₂—), 4.525 (m, 2H), 3.936 (m, 2H), 3.834 (dd, 1H,J=3.8, J=9.9), 3.500, 3.397 (s, 3H, CH ₃OCH₂—)×2, 2.105, 2.090 (s, 3H,CH ₃C═O)×2

(16) Synthesis of Compound 20

Compound 19 (8.96 g, 24.0 mmol) was dissolved in methanol (300 ml), andsodium methoxide (262.7 mg, 4.863 mmol) was added thereto, followed bystirring at room temperature for 80 minutes. After completion of thereaction, the reaction solution was neutralized with Amberlist 15 DRY(trade name, manufactured by Organo Corporation) and filtered throughcelite. The solvent was evaporated under reduced pressure, and theresulting residue was purified by silica gel column chromatography(chloroform methanol=40: 1, 0.2% triethylamine) to obtain Compound 20(5.11 g, 73.6%).

TLC: Rf=0.33 (toluene:acetone=1:1)

¹H-NMR (500 MHz, CDCl₃)

δ=5.727 (m, 1H, H-5a), 4.889 (d, 1H, J=6.6, CH₃OCH ₂—), 4.845 (d, 1H,J=6.8, CH₃OCH ₂—), 4.790 (d, 1H, J=6.6, CH₃OCH ₂—), 4.786 (d, 1H, J=6.6,CH₃OCH ₂—), 4.358 (br.t, J=3.1), 4.245 (br.m, 2H), 3.970 (dd, 1H, J=7.5,J=9.6), 3.874 (m, 1H), 3.700 (dd, 1H, J=3.9, J=9.8), 3.477, 3.444 (s,3H, CH ₃OCH₂—)×2, 3.099 (br, 1H), 2.527 (br, 1H)

(17) Synthesis of Compound 21

In argon atmosphere, N-ethyldiisopropylamine (61.5 ml, 353.06 mmol) wasadded to a dichloroethane solution (300 ml) of Compound 20 (5.11 g, 17.7mmol), and chloromethyl methyl ether (13.4 ml, 176.4 mmol) was addeddropwise thereto, followed by stirring at room temperature for 15minutes and then at 60° C. for 5.5 hours. The reaction solution wasice-cooled, triethylamine (60 ml) was added thereto, and ethanol (60 ml)was added dropwise thereto, followed by stirring at room temperature for35 minutes. The solvent in the reaction solution was evaporated underreduced pressure, the resulting residue was dissolved in ethyl acetateand washed with saturated sodium bicarbonate water and saturated brinein this order, the resulting organic layer was dried over magnesiumsulfate, and the solvent was evaporated under reduced pressure. Theresulting residue was purified by silica gel column chromatography(toluene:ethyl acetate=3:1, 0.2% triethylamine) to obtain Compound 21(6.65 g, 99.8%).

TLC: Rf=0.44 (toluene ethyl acetate=1:1)

¹H-NMR (500 MH, CDCl₃)

δ=5.740 (m, 1H, H-5a), 4.926 (d, 1H, J=6.4, CH₃OCH ₂—), 4.874 (d, 1H,J=6.8, CH₃OCH ₂), 4.776 (m, 3H, CH₃OCH ₂—×3), 4.684 (d, 1H, J=7.1,CH₃OCH ₂—) 4.651 (d, 2H, J=6.6, CH₃OCH ₂—×2), 4.218 (d, 1H, J=3.2, H-4),4.118 (m, 2H, 4.079 (dd, 1H, J=7.7, J=10.1, H-2), 3.846 (br.d, 1H),3.721 (dd, 1H, J=3.3, J=10.1, H-3), 3.485, 3.422, 3.398, 3.385 (s, 3H,CH ₃OCH₂—)×4

(18) Synthesis of Compound 22

Water (2.5 ml) and triphenylphosphine (404.1 mg, 1.54 mmol) were addedto a toluene solution (10 ml) of Compound 21 (290.4 mg, 0.769 mmol),followed by stirring at 105° C. for 105 minutes. Toluene and ethanolwere added to the reaction solution, evaporation of the solvent underreduced pressure was repeated 3 times, and the resulting residue waspurified by silica gel column chromatography (chloroform:methanol=20:1,0.5% triethylamine) to obtain Compound 22 (267.3 mg, 99%).

TLC: Rf=0.47 (chloroform:methanol=10:1)

¹H-NMR (500 MHz, CDCl₃)

δ=5.754 (m, 1H), 4.903 (d, 1H, J=6.8, CH₃OCH ₂—), 4.898 (d, 1H, J=6.6,CH₃OCH ₂—), 4.767 (m, 3H, CH₃OCH ₂—×3), 4.695 (d, 1H, J=6.8, CH₃OCH ₂—),4.658 (d, 1H, J=6.6, CH₃OCH ₂—), 4.638 (d, 1H, J=6.6, CH₃OCH ₂—), 4.252(d, 1H, J=2.7, H-4), 4.124 (dt, 1H, J=1.7, J=12.2), 4.039 (dtm, 1H,J=1.0, J=12.2), 3.704 (m, 2H), 3.455, 3.420, 3.410, 3.385 (s, 3H, CH₃OCH₂—)×4

(19) Synthesis of Compound 23

In argon atmosphere, triethylamine (4.13 ml, 29.6 mmol) was added to adichloromethane solution (50 ml) of Compound 22 (2.60 g, 7.40 mmol),followed by ice-cooling, and di-t-butyl dicarbonate (3.40 ml, 14.8 mmol)was added thereto, followed by stirring for 80 minutes while graduallyraising the temperature of the reaction solution to room temperature.The reaction solution was ice-cooled, diluted with ethyl acetate andwashed with saturated sodium bicarbonate water and saturated brine inthis order, the resulting organic layer was dried over magnesiumsulfate, and the solvent was evaporated under reduced pressure. Theresulting residue was purified by silica gel column chromatography(toluene:ethyl acetate=3:2, 0.5% triethylamine) to obtain Compound 23(3.31 g, 99%).

TLC: Rf=0.25 (toluene:ethyl acetate=1:1)

¹H-NMR (500 MHz, CDCl₃)

δ=5.752 (br.s, 1H), 4.911 (br.d, 1H, J=8.6), 4.862 (d, 1H, J=6.8, CH₃OCH₂—), 4.833 (d, 1H, J=6.8, CH₃OCH ₂—), 4.780 (d, 1H, J=6.6, CH₃OCH ₂—),4.749 (d, 1H, J=6.6, CH₃OCH ₂—), 4.693 (d, 1H, J=6.8, CH₃OCH ₂—), 4.690(d, 1H, J=6.8, CH₃OCH ₂—), 4.645 (d, 1H, J=6.6, CH₃OCH ₂—), 4.623 (d,1H, J=6.6, CH₃OCH ₂—), 4.295 (d, 1H, J=2.0, H-4), 4.241 (br, 1H), 4.119(br.d, 1H, J=12.7), 4.035 (br.d, 1H, J=12.5), 3.854 (m, 2H), 3.413,3.405, 3.400, 3.371 (s, 3H, CH ₃OCH₂—)×4

(20) Synthesis of Compound 24-1

In argon atmosphere, a DMP solution (1.5 ml) of Compound 23 (100 mg,0.221 mmol) was ice-cooled, and sodium hydride (60% in oil, 31.8 mg,0.795 mmol) was added thereto, followed by stirring for 10 minutes underice-cooling, Then, bromobutane (31.6 μl, 0.831 mmol) was added thereto,followed by stirring for 4 hours while gradually raising the temperatureof the reaction solution to room temperature. The reaction solution wasice-cooled, and methanol (1 ml) was added dropwise thereto, followed bystirring for 30 minutes. The reaction solution was diluted with ethylacetate and stirred by adding saturated sodium bicarbonate water, theorganic layer was washed with saturated sodium bicarbonate water andsaturated brine in this order and dried over magnesium sulfate, and thesolvent was evaporated under reduced pressure. The resulting residue waspurified by silica gel column chromatography (toluene:ethyl acetate 2:1,0.2% triethylamine) to obtain Compound 24-1 (102.3 mg, 91.0%).Developing solvent of Compound 241 (toluene; ethyl acetate=2:1) Rf: 0.32

C₂₄H₄₅NO₁₀ MW: 507.61

¹H-NMR (500 MHz, CDCl₃)

δ=5.574 (br.d, 1H, H-5a), 4.910, 4.871 (2d, 2H, J=6.8 Hz, 6.8 Hz, OCH₂),4.801-4.618 (m, 6H, 3OCH₂), 4.188 (br.d, 1H), 4.112-4.039 (m, 3H), 3.737(br.d, 1H), 3.406, 3.387, 3.368, 3.348 (4s, 12H, 4OCH₃), 3.135 (m, 1H,NCH₂), 2.907 (m, 1H, NCH₂), 1.66-1.22 (m, 4H, 2CH₂), 1.464 (s, 9H,CC(CH₃)₃), 0.892 (t, 3H, J=7.3 Hz, CH₃)

(21) Synthesis of Compound 25-1

Hydrochloric acid (4 N, 6.5 ml) was added to a THF solution (4.0 ml) ofCompound 24-1 (102 mg, 0.201 mmol), followed by stirring at 45° C. for 5hours. The solvent of the reaction solution was evaporated under reducedpressure, and the resulting residue was azeotroped with ethanol 3 times.The resulting residue was purified by Sephadex LH-20 (trade name,manufactured by Pharmacia Biotech) (chloroform:methanol=1:2). Then, themixture was dissolved in water (1 ml), 25% aqueous ammonia (1 ml) wasadded thereto, and the reaction solution was directly purified by silicagel column chromatography (chloroform:methanol:water=60:35:8) to obtainCompound 25-1 (15 mg, 32.3%).

Developing solvent of Compound 25-1 (chloroform:methanol:water=60:35:8)Rf: 0.15

C₁₁H₂₁NO₄ MW: 231.29

¹H-NMR (500 MHz, CD₃OD)

δ=5.723 (d, 1H, J=2.0 Hz, H-5a), 4.155 (d, 1H, J=4.2 Hz, H-4), 4.128(br.s, 2H, H-6), 3.726 (dd, 1H, J=8.3 Hz, 10.0 Hz, H-2), 3.447 (dd, 1H,J=J=4.2 Hz, 10.1 Hz, H-3), 3.169 (br.d, 1H, H-1), 2.756 (br.ddd, 1H,NCH₂), 2.621 (br.ddd, 1H, NCH₂), 1.56-1.28 (m, 4H, 2CH₂), 0.953 (t, 3H,3=7.3 Hz, CH₃)

(22) Synthesis of Compound 24-2

In argon atmosphere, dimethylformamide solution (50 ml) of Compound 23(3.31 g, 7.33 mmol) was ice-cooled, and sodium hydride (60% in oil,1051.0 mg, 26.3 mmol) was added thereto, followed by stirring for 10minutes under ice-cooling. Then, n-octyl bromide (1.90 ml, 10.99 mmol)was added dropwise thereto, followed by stirring for 140 minutes whilegradually raising the temperature of the reaction solution to roomtemperature. The reaction solution was ice-cooled and methanol (1.78 ml)was added dropwise thereto, followed by stirring for 30 minutes. Thereaction solution was diluted with diethyl ether (200 ml) and saturatedsodium bicarbonate water (100 ml) was added thereto, followed bystirring. Then, the organic layer was washed with saturated sodiumbicarbonate water and saturated brine in this order and dried overmagnesium sulfate, and the solvent was evaporated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (toluene ethyl acetate=3:1, 0.5% triethylamine) to obtainCompound 24-2 (3.62 g, 88%).

Developing solvent of Compound 24-2 (toluene:ethyl acetate=1:1) Rf: 0.40

¹H-NMR (500 MHz, CDCl₃)

C₂₈H₅₃NO₁₀ MW: 563.72

δ=5.573 (br.s, 1H), 4.909 (d, 1H, J=6.8, CH₃OCH ₂—), 4.873 (d, 1H,J=6.8, CH₃OCH ₂—), 4.794 (br.d, 1H, J=6.6, CH₃OCH ₂—), 4.740 (d, 1H,J=6.4, CH₃OCH ₂—), 4.699 (d, 1H, J=6.8, CH₃OCH ₂—), 4.633 (m, 3H, CH₃OCH₂—×3), 4.190 (br.s, 1H), 4.089 (m, 3H), 3.736 (br.d, 1H, J=10.3), 3.406,3.390, 3.366, 3.345 (s, 3H, CH ₃OCH₂—)×4, 3.121 (br, 1H), 2.894 (br,1H), 1.462 (br×2, 9H, t-butyl), 1.261 (br.m, 12H, H-2′-7′), 0.878 (t,3H, H-8′)

(23) Synthesis of Compound 25-2

Hydrochloric acid (4 N, 187 ml) was added to a THF solution (123 ml) ofCompound 24-2 (3.85 g, 6.83 mmol), followed by stirring at 45° C. for 3hours. The solvent of the reaction solution was evaporated under reducedpressure, and the resulting residue was azeotroped with 300 ml ofethanol 3 times. The resulting residue was purified by Sephadex LH-20(trade name, manufactured by Pharmacia Biotech)(chloroform:methanol=1:1). Then, the mixture was dissolved in water (200ml), and 25% aqueous ammonia (10 ml) was added thereto. Ten minutesthereafter, the precipitated crystals were collected by filtration toobtain Compound 25-2 (1.35 g, 72.1%).

Melting point: 80.9-82.3° C.

[α]²⁰ _(D) =+9.83° (c=1.0 methanol)

¹H-NMR (500 MHz, 1:2=CD₃OD:CDCl₂)

δ=5.734 (br.d, 1H, H-5a), 4.268 (br.d, 1H, H-6a), 4.153 (d, 1H, J=4.1Hz, H-1), 4.133 (br.d, 1H, H-6b), 3.953 (dd, 1H, J=8.1, 9.8 Hz, H-3),3.638 (br.d, 1H, H-4), 3.549 (dd, 1H, J=4.1, 9.8 Hz, H-2), 3.063 (m, 2H,H-1′×2), 1.75 1.26 (m, 12H, 6CH₂, H-2′-7′), 0.894 (t, 3H, J=7.1 Hz, CH₃,H-8′)

(24) Synthesis of Compound 243

In argon atmosphere, a DMF solution (2 ml) of Compound 23 (85 mg, 0.188mmol) was ice-cooled, and sodium hydride (60% in oil, 26.7 mg, 0.668mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, 1-bromodecane (58.6 μl, 0.283 mmol) was addeddropwise thereto, followed by stirring for 3 hours while graduallyraising the temperature of the reaction solution to room temperature.The reaction solution was ice-cooled, and ethanol (1 ml) was addeddropwise thereto, followed by stirring for 30 minutes. The reactionsolution was diluted with ethyl acetate, and saturated sodiumbicarbonate water was added thereto, followed by stirring. Then, theorganic layer was washed with saturated sodium bicarbonate water andsaturated brine in this order and dried over magnesium sulfate, and thesolvent was evaporated under reduced pressure. The resulting residue waspurified by silica gel column chromatography (toluene:ethyl acetate=4:1,0.2% triethylamine) to obtain Compound 24-3 (91.5 mg, 82.1%).

Developing solvent of Compound 24-3 (toluene:ethyl acetate=4:1) Rf: 0.27

C₃₀H₅₇NO₁₀ MW: 591.78

¹H-NMR (500 MHz, CDCl₃)

δ=5.574 (br.d, 1H, H-5a), 4.907, 4.873 (2d, 2H, J=6.6 Hz, 6.7 Hz, OCH₂),4.800-4.617 (m, 6H, 3OCH₂), 4.191 (br.d, 1H), 4.113-4.035 (m, 3H), 3.736(br.d, 1H), 3.406, 3.389 3.366, 3.345 (4s, 12H, 4OCH₃), 3.121 (n, 1H,NCH₂), 2.895 (m, 1H, NCH₂), 1.67-1.25 (m, 16H, 3OCH₂), 1.251 (s, 9H,CC(CH₃)₃), 0.880 (t, 3H, J=7.1 Hz, CH₃)

(25) Synthesis of Compound 25-3

Hydrochloric acid (4 N, 6.5 ml) was added to a THF solution (4 ml) ofCompound 24-3 (91.5 mg, 0.155 mmol), followed by stirring at 45° C. for5 hours. The solvent of the reaction solution was evaporated underreduced pressure, and the resulting residue was azeotroped with ethanol3 times. The resulting residue was purified by Sephadex LH-20 (tradename, manufactured by Pharmacia Biotech)(chloroform:methanol:water=1:2). Then, the mixture was dissolved inwater (1 ml), and 25% aqueous ammonia (1 ml) was added thereto. Tenminutes thereafter, the precipitated crystals were collected byfiltration to obtain Compound 25-3 (41 mg, 83.9%).

Developing solvent of Compound 25-3 (chloroform:methanol:water=60:35:8)Rf: 0.31

C₁₇H₃₃NO₄ MW: 315.45

¹H-NMR (500 MHz, CD₃OD)

δ=5.727 (d, 1H, J=2.2 Hz, H-5a), 4.167 (d, 1H, J=4.2 Hz, H-4), 4.143(br.s, 2H, H-6), 3.684 (dd, 1H, J=8.3 Hz, 10.0 Hz, H-2), 3.463 (dd, 1H,J=4.2 Hz, 10.3 Hz, H-3), 3.124 (br.d, 1H, H-1), 2.762 (br.ddd, 1H,NCH₂), 2.571 (br.ddd, 1H, NCH₂), 1.55-1.29 (m, 16H, 8CH₂), 0.893 (t, 3H,J=7.0 Hz, CH₃)

(26) Synthesis of Compound 24-4

In argon atmosphere, a DMF solution (2 ml) of Compound 23 (84 mg, 0.1860mmol) was ice-cooled, and sodium hydride (60% in oil, 26.7 mg, 0.925mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Lauryl bromide (47.8 μl, 0.279 mmol) was added dropwisethereto, followed by stirring for 3.5 hours while gradually raising thetemperature of the reaction solution to room temperature. The reactionsolution was ice-cooled, and methanol (1 ml) was added dropwise thereto,followed by stirring for 30 minutes. The reaction solution was dilutedwith ethyl acetate, and saturated sodium bicarbonate water was addedthereto, followed by stirring. Then, the organic layer was washed withsaturated sodium bicarbonate water and saturated brine in this order anddried over magnesium sulfate, and the solvent was evaporated underreduced pressure. The resulting residue was purified by silica gelcolumn chromatography (toluene:ethyl acetate=3:1, 0.2% triethylamine) toobtain Compound 24-4 (86.9 mg, 75.4%).

Developing solvent of Compound 24-4 (toluene:ethyl acetate=2:1) Rf: 0.33

C₃₂H₆₁NO₁₀ MW: 619.83

¹H-NMR (500 MHz, CDCl₃)

δ=5.573 (br.d, 1H H-5a), 4.909, 4.873 (2d, 2H, J=6.8 Hz, 6.7 Hz, OCH₂),4.801-4.616 (m, 6H, 3OCH₂), 4.190 (br.d, 1H), 4.112-4.035 (m, 3H), 3.736(br.d, 1H), 3.406, 3.389, 3.365, 3.345 (4s, 12H, 4OCH₃), 3.121 (m, 1H,NCH₂), 2.891 (m, 1H, NCH₂), 1.65-1.25 (m, 20H, 10CH₂), 1.250 (s, 9H,CC(CH₃)₃), 0.881 (t, 3H, J=7.1 Hz, CH₃)

(27) Synthesis of Compound 25-4

Hydrochloric acid (4 N, 4.6 ml) was added to a THF solution (3 ml) ofCompound 24-4 (86.9 mg, 0.140 mmol), followed by stirring at 45° C. for5 hours. The solvent of the reaction solution was evaporated underreduced pressure, and the resulting residue was azeotroped with ethanol3 times. The resulting residue was purified by Sephadex LH-20 (tradename, manufacured by Pharmacia Biotech) (chloroform:methanol=1:2). Then,the mixture was dissolved in water (1 ml), and 25% aqueous ammonia (1ml) was added thereto. Ten minutes thereafter, the precipitated crystalswere collected by filtration to obtain Compound 25-4 (41.1 mg, 85.5%).

Developing solvent of Compound 25-4 (chloroform:methanol:water=60:35:8)Rf: 0.31

C₁₉H₃₇NO₄ MW: 343.50

¹H-NMR (500, MHz, CD₃OD)

δ=5.726 (d, 1H, J=2.2 Hz, H-5a), 4.165 (d, 1H, J=4.4 Hz, H-4), 4.138(br.s, 2H, H-6), 3.682 (dd, 1H, J=8.1 Hz, 10.3 Hz, H-2), 3.457 (dd, 1H,J=4.2 Hz, 10.3 Hz, H-3), 3.110 (br.d, 1H, H-1), 2.752 (br.ddd, 1H,NCH₂), 2.558 (br.ddd, 1H, NCH₂), 1.55-1.28 (m, 20H, 10CH₂), 0.893 (t,3H, J=7.1 Hz, CH₃)

(28) Synthesis of Compound 24-5

In argon atmosphere, a DMF solution (1.5 ml) of Compound 23 (70 mg,0.155 mmol) was ice-cooled, sodium hydride (60% in oil, 22.2 mg, 0.555mmol) was added thereto, followed by stirring for 10 minutes underice-cooling, and myristyl bromide (63 μl, 0.233 mmol) was added dropwisethereto, followed by stirring for 2.5 hours while gradually raising thetemperature of the reaction solution to room temperature. The reactionsolution was ice-cooled, and methanol (1 ml) was added dropwise thereto,followed by stirring for 30 minutes. The reaction solution was dilutedwith ethyl acetate, and saturated sodium bicarbonate water was addedthereto, followed by stirring. Then, the organic layer was washed withsaturated sodium bicarbonate water and saturated brine in this order anddried over magnesium sulfate, and the solvent was evaporated underreduced pressure. The resulting residue was purified by silica gelcolumn chromatography (toluene:ethyl acetate=3:1, 0.2% triethylamine) toobtain Compound 24-5 (85.6 mg, 85.2%).

Developing solvent of Compound 24-5 (toluene: ethyl acetate=3:1) Rf:0.34

C₃₄H₆₅NO₁₀ MW: 647.88

¹H-NMR (500 MHz, CDCl₃)

δ=5.571 (br.d, 1H, H-5a), 4.907, 4.873 (2d, 2H, J=6.6 Hz, 6.8 Hz, OCH₂),4.800-4.616 (m) 6H, 3OCH₂), 4.189 (br.d, 1H), 4.112-4.034 (m, 3H), 3.735(br.d, 1H), 3.405, 3.388, 3.364, 3.344 (4s, 12H, 4OCH₃), 3.119 (m, 1H,NCH₂), 2.892 (m, 1H, NCH₂), 1.67-1.25 (m, 24H, 12CH₂), 1.251 (s, 9H,CC(CH₃)₃), 0.880 (t 3H, J=7.1 Hz, CH₃)

(29) Synthesis of Compound 25-5

Hydrochloric acid (4 N, 4 ml) was added to a THF solution (3 ml) ofCompound 24-5 (85.6 mg, 0.132 mmol), followed by stirring at 45° C. for5 hours. The solvent of the reaction solution was evaporated underreduced pressure, and the resulting residue was azeotroped with ethanol3 times. The resulting residue was purified by Sephadex LH-20 (tradename, manufactured by Pharmacia Biotech) (chloroform methanol=1:2).Then, the mixture was dissolved in water (1 ml), and 25% aqueous ammonia(1 ml) was added thereto, Ten minutes thereafter, the precipitatedcrystals were collected by filtration to obtain Compound 25-5 (48.1 mg,98-1%).

Developing solvent of Compound 25-5 (chloroform:methanol:water=60:35:8)Rf: 0.38

C₂₁H₄₁NO₄ MW: 371.55

¹H-NMR (500 MHz, CD₃OD)

δ=5.726 (d, 1H, J=2.0 Hz, H-5a), 4.165 (d, 1H, J=4.2 Hz, H-4), 4.138(br.s, 2H, H-6), 3.683 (dd, 1H, J=8.1 Hz, 10.3 Hz, H-2), 3.457 (dd, 1H,J=4.2 Hz, 10.3 Hz, H-3), 3.110 (br.d, 1H, H-1), 2.752 (br.ddd, 1H,NCH₂), 2.559 (br.ddd, 1H, NCH₂), 1.56-1.28 (m, 24H, 12CH₂), 0.894 (t,3H, J=7.1 Hz, CH₃)

(30) Synthesis of Compound 24-6

In argon atmosphere, a DMF solution (1.5 ml) of Compound 23 (70 mg,0.155 mmol) was ice-cooled, and sodium hydride (60% in oil, 22.2 mg,0.555 mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, 1-bromooctadecane (77.5 mg, 0.232 mmol) was addedthereto, followed by stirring for 4 hours while gradually raising tiletemperature of the reaction solution to room temperature. The reactionsolution was ice-cooled, and methanol (1 ml) was added dropwise thereto,followed by stirring for 30 minutes. The reaction solution was dilutedwith ethyl acetate, and saturated sodium bicarbonate water was addedthereto, followed by stirring. Then, the organic layer was washed withsaturated sodium bicarbonate water and saturated brine in this order anddried over magnesium sulfate, and the solvent was evaporated underreduced pressure. The resulting residue was purified by silica gelcolumn chromatography (toluene:ethyl acetate=3:1, 0.2% triethylamine) toobtain Compound 24-6 (57.5 mg, 52.7%).

Developing solvent of Compound 24-6 (toluene: ethyl acetate=2:1) Rf 0.36C₃₈H₇₃NO₁₀ MW: 703.99

¹H-NMR (500 MHz, CDCl₃)

δ=5.572 (br.d, 1H, H-5a), 4.909, 4.873 (2d, 2H, J=6.8 Hz, 6.8 Hz, OCH₂),4.801-4.616 (m, 6H, 3OCH₂), 4.190 (br.d, 1H), 4.113-4.035 (m, 3H), 3.739(br.d, 1H), 3.406, 3.389, 3.365, 3.345 (4s, 12H, 4OCH₃), 3.121 (m, 1H,NCH₂), 2.887 (m, 1H, NCH₂), 1.67-1.25 (m, 32H, 16CH₂), 1.255 (s, 9H,CC(CH₃)₃), 0.880 (t, 3H, J=7.1 Hz, CH₃)

(31) Synthesis of Compound 25-6

Hydrochloric acid (4 N, 2.5 ml) was added to a THF solution (2.0 ml) ofCompound 24-6 (57.5 mg, 0.082 mmol), followed by stirring at 45° C. for5 hours. The solvent of the reaction solution was evaporated underreduced pressure, and the resulting residue was azeotroped with ethanol3 times. The resulting residue was purified by Sephadex LH-20 (tradename, manufactured by Pharmacia Biotech) (chloroform:methanol=1:2).Then, the mixture was dissolved in water (1 ml), and 25% aqueous ammonia(1 ml) was added thereto. Ten minutes thereafter, the precipitatedcrystals were collected by filtration to obtain Compound 25-6 (26.9 mg,76.7%).

Developing solvent of Compound 25-6 (chloroform:methanol:water 60:35:8)Rf: 0.43

C₂₅H₄₉NO₄ MW: 427.66

¹H-NMR (500 MHz, CD₃OD)

δ=5.729 (d, 1H, J=1.8 Hz, H-5a), 4.168 (d, 1H, J=4.0 Hz, H-4), 4.145(br.s, 2H, H-6), 3.675 (dd, 1H, J=8.3 Hz, 10.3 Hz, H-2), 3.463 (dd, 1H,J=4.1 Hz, 10.3 Hz, H-3), 3.109 (br.d, 1H, H-1), 2.754 (br.ddd, 1H,NCH₂), 2.557 (br.ddd, 1H, NCH₂), 1.53-1.27 (m, 32H, 16CH₂), 0.893 (t,3H, J=7.0 Hz, CH₃)

(32) Synthesis of Compound 24-7

In argon atmosphere, a DMF solution (2.0 ml) of Compound 23 (85 mg,0.188 mmol) was ice-cooled, and sodium hydride (60% in oil, 27.0 mg,0.675 mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, bromodocosane (110 mg, 0.282 mmol) was added thereto,followed by stirring for 2 hours while gradually raising the temperatureof the reaction solution to room temperature. The reaction solution wasice-cooled, and methanol (1 ml) was added dropwise thereto, followed bystirring for 30 minutes. The reaction solution was diluted with ethylacetate, and saturated sodium bicarbonate water was added thereto,followed by stirring. Then, the organic layer was washed with saturatedsodium bicarbonate water and saturated brine in this order and driedover magnesium sulfate, and the solvent was evaporated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (toluene:ethyl acetate=3:1, 0.2% triethylamine) to obtainCompound 24-7 (70.2 mg, 49.1%).

Developing solvent of Compound 24-7 (toluene:ethyl acetate=2:1) Rf: 0.38

C₄₂H₈₁NO₁₀ MW: 760.10

¹H-NMR (500 MHz, CDCl₃)

δ=5.572 (br.d, 1H, H-5a), 4.909, 4.873 (2d, 2H, J=6.6 Hz, 6.8 Hz, OCH₂),4.801-4.616 (m, 6H 3OCH₂), 4.190 (br.d, 1H), 4.112-4.035 (m, 3H), 3.736(br.d, 1H), 3.406, 3.389, 3.365, 3.345 (4s, 12H, 4OCH₃), 3.119 (m, 1H,NCH₂), 2.887 (m, 1H, NCH₂), 1.67-1.25 (m, 40H, 20CH₂), 1.254 (s, 9H,CC(CH₃)₃), 0.880 (t, 3H, J=7.1 Hz, CH₃)

(33) Synthesis of Compound 25-7

Hydrochloric acid (4 N. 3.0 ml) was added to a THF solution (2.0 ml) ofCompound 24-7 (70.2 mg, 0.092 mmol), followed by stirring at 45° C. for5 hours. The solvent of the reaction solution was evaporated underreduced pressure, and the resulting residue was azeotroped with ethanol3 times. The resulting residue was purified by Sephadex LH-20 (tradename, manufactured by Pharmacia Biotech) (chloroform:methanol=1:2).Then, the mixture was dissolved in water (1 ml), and 25% aqueous ammonia(1 ml) was added thereto. Ten minutes thereafter, the precipitatedcrystals were collected by filtration to obtain Compound 25-7 (27.1 mg,60.9%).

Developing solvent of Compound 25-7 (chloroform methanol:water=60:35:8)Rf: 0.48

C₂₉H₅₇NO₄ MW: 483.77

¹H-NMR (500 MHz, CD₃OD)

δ=5.727 (d, 1H, J=1.8 Hz, H-5a), 4.166 (d, 1H) J=4.2 Hz, H-4), 4.140(br.s, 2H, H-6), 3.683 (dd, 1H, J=8.4 Hz, 10.3 Hz, H-2), 3.460 (dd, 1H,J=J=4.1 Hz, 10.2 Hz, H-3), 3.113 (br.d, 1H, H-1), 2.756 (br.ddd, 1H,NCH₂), 2.563 (br.ddd, 1H, NCH₂), 1.53-1.24 (m, 40H, 20CH₂), 0.893 (t,3H, J=7.1 Hz, CH₃)

(34) Synthesis of Compound 26

In argon atmosphere, N-ethyldiisopropylamine (322 μl, 20 eq) was addedto a dichloroethane solution (3 ml) of Compound 17 (26.7 mg, 0.0922mmol), and chloromethyl methyl ether (70.1 μl, 10 eq) was added dropwisethereto, followed by stirring at room temperature for 15 minutes andthen at 60° C. for 1 day. The reaction solution was ice-cooled,triethylamine (1 ml) was added thereto, and ethanol (1 ml) was addeddropwise thereto, followed by stirring at room temperature for 35minutes. The solvent of the reaction solution was evaporated underreduced pressure, the resulting residue was dissolved in ethyl acetateand washed with saturated sodium bicarbonate water and saturated brinein this order, the resulting organic layer was dried over magnesiumsulfate, and the solvent was evaporated under reduced pressure. Theresulting residue was purified by silica gel column chromatography(toluene:ethyl acetate=1:1, 0.2% triethylamine) to obtain Compound 26(34 mg, 97.6%).

Developing solvent of Compound 26 (toluene:ethyl acetate=1:1) Rf: 0.44

C₁₅H₂₇N₃O₈ MW: 377.39

¹H-NMR (500 MHz, CDCl₃)

δ=5.755 (br.s, 1H, H-5a), 4.899, 4.894, 4.854, 4.812, 4.781, 4.729,4.668, 4.650 (8d, 8H, J=6.6 Hz, 6.6 Hz, 6.6 Hz, 6.4 Hz, 6.6 Hz, 6.6 Hz,6.4 Hz, 6.6 Hz, 4OCH₂), 4.203 (br.d, 1H), 4.152 (br.s, 2H), 3.964 (n,1H), 3.833 (dd, 1H, J=7.1 Hz, 9.0 Hz), 3.701 (dd, 1H, J=7.3 Hz, 9.0 Hz),3.476, 3.440, 3.380 (3s, 12H, 4OCH₃)

(35) Synthesis of Compound 27

Water (30 ml) and triphenylphosphine (6.03 g, 2.0 eq) were added to atoluene solution (150 ml) of Compound 26 (4.34 g, 11.5 mmol), followedby stirring at 105° C. for 105 minutes. Toluene and ethanol were addedto the reaction solution, evaporation of the solvent under reducedpressure was repeated 3 times, and the resulting residue was purified bysilica gel column chromatography (chloroform:methanol=20:1, 0.2%triethylamine) to obtain Compound 27 (3.86 g, 95.5%).

Developing solvent of Compound 27 (chloroform:methanol=10:1) Rf: 0.35C₁₅H₂₉NO_(g) MW; 351.39

¹H-NMR (500 MHz, CDCl₃)

δ=5.743 (br.s, 1H, H-5a), 4.896, 4.854, 4.833, 4.811, 4.765, 4.744,4.663, 4.644 (8d, 8H, J=6.6 Hz, 6.8 Hz, 6.3 Hz, 6.6 Hz, 6.8 Hz, 6.6 Hz,6.6 Hz, 6.6 Hz, 4OCH₂), 4.212 (br.d, 1H), 4.148, 4.078 (2br.d, 2H, H-6a,H-6b), 3.837 (m, 1H), 3.443, 3.439, 3.378 (3s, 12H, 4OCH₃)

(36) Synthesis of Compound 28

In argon atmosphere, triethylamine (211 μl, 1.515 mmol) was added to adichloromethane solution (3 ml) of Compound 27 (131.1 mg, 0.379 mmol),followed by ice-cooling, and di-t-butyl dicarbonate (174 μl, 0.758 mmol)was added thereto, followed by stirring for 50 minutes while graduallyraising the temperature of the reaction solution to room temperature.The reaction solution was ice-cooled, diluted with ethyl acetate andwashed with saturated sodium bicarbonate water and saturated brine inthis order, the resulting organic layer was dried over magnesiumsulfate, and the solvent was evaporated under reduced pressure. Theresulting residue was purified by silica gel column chromatography(toluene:ethyl acetate=3:2, 0.2% triethylamine) to obtain Compound 28(168.6 mg, 98.6%).

Developing solvent of Compound 28 (toluene:ethyl acetate=1:1) Rf: 0.30

C₂₀H₃₇NO₁₀ MW: 451.51

¹H-NMR (500 MHz, CDCl₃)

δ=5.843 (br.s, 1H, H-5a), 4.982 (d, 1H, J=9.2 Hz, NH), 4.868, 4.790,4.785, 4.769, 4.736, 4.723, 4.655, 4.633 (8d, 8H, J=6.8 Hz, 6.6 Hz, 7.1Hz, 6.6 Hz, 6.6 Hz, 6.1 Hz, 6.6 Hz, 6.6 Hz, 4OCH₂), 4.291 (br.d, 1H),4.177-4.145 (m, 2H), 4.077 (br.d, 1H), 3.990 (br.dd, 1H), 3.693 (m, 1H),3.443, 3.422, 3.410, 3.375 (4s, 12H, 4OCH₃), 1.436 (s, 9H, CC(CH₃)₃)

(37) Synthesis of Compound 29-1

In argon atmosphere, a DMF solution (1.5 ml) of Compound 28 (88.2 mg,0.195 mmol) was ice-cooled, and sodium hydride (60% in oil, 28.1 mg,0.703 mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, bromobutane (31.5 μl, 0.293 mmol) was added thereto,followed by stirring for 4 hours while gradually raising the temperatureof the reaction solution to room temperature. The reaction solution wasice-cooled, and methanol (1 ml) was added dropwise thereto, followed bystirring for 30 minutes. The reaction solution was diluted with diethylether, and saturated sodium bicarbonate water was added thereto,followed by stirring. Then, the organic layer was washed with saturatedsodium bicarbonate water and saturated brine in this order and driedover magnesium sulfate, and the solvent was evaporated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (toluene:ethyl acetate=10:1, 0.2% triethylamine) toobtain Compound 29-1 (94.4 mg, 95.2%).

Developing solvent of Compound 29-1 (toluene:ethyl acetate=1:1) Rf: 0.63

C₂₄H₄₅NO₁₀ MW: 507.61

(38) Synthesis of Compound 30-1

Hydrochloric acid (4 N, 5.0 ml) was added to a THF solution (3.0 ml) ofCompound 29-1 (94.4 mg, 0.186 mmol), followed by stirring at roomtemperature for 1 day. The solvent of the reaction solution wasevaporated under reduced pressure, and the resulting residue wasazeotroped with ethanol 3 times. The resulting residue was purified bysilica gel column chromatography (chloroform:methanol:water=60:35:8).Then, the mixture was dissolved in methanol (0.5 ml)-water (0.5 ml), and25% aqueous ammonia (0.5 ml) was added thereto. The reaction solutionwas directly purified by silica gel column chromatography(chloroform:methanol:water=60:35:8) to obtain Compound 30-1 (37.2 mg,86.5%).

Developing solvent of Compound 30-1 (chloroform:methanol:water 60:35:8)Rf: 0.18

C₁₁H₂₁NO₄ MW: 231.29

¹H-NMR (500 MHz, CDCl₃:CD₃OD=2:1)

δ=5.615 (br.s, 1H, H-5a), 4.194, 4.114 (br.2d, 2H, H-6a, H-6b), 4.156(m, 1H), 3.553 (dd, 1H, J=7.6 Hz, 10.0 Hz), 3.445 (dd, 1H, J=8.3 Hz,10.0 Hz), 3.260 (br.d, 1H), 2.781 (br.ddd, 1H, NCH₂), 2.590 (br.ddd, 1H,NCH₂), 1.55-1.26 (m, 4H, 2CH₂), 0.950 (t, 3H, J=7.3 Hz, CH₃)

(39) Synthesis of Compound 29-2

In argon atmosphere, a DMF solution (3 ml) of Compound 28 (168.6 mg,0.373 mmol) was ice-cooled, and sodium hydride (60% in oil, 53.8 mg,1.344 mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, n-octyl bromide (96.8 μl, 0.560 mmol) was addeddropwise thereto, followed by stirring for 3 hours while graduallyraising the temperature of the reaction solution to room temperature,The reaction solution was ice-cooled, and methanol (1 ml) was addeddropwise thereto, followed by stirring for 30 minutes. The reactionsolution was diluted with diethyl ether (200 ml), and saturated sodiumbicarbonate water was added thereto, followed by stirring. Then, theorganic layer was washed with saturated sodium bicarbonate water andsaturated brine in this order and dried over magnesium sulfate, and thesolvent was evaporated under reduced pressure. The resulting residue waspurified by silica gel column chromatography (toluene:ethyl acetate=3:1,0.2% triethylamine) to obtain Compound 29-2 (155.1 mg, 73.7%).

Developing solvent of Compound 29-2 (toluene:ethyl acetate=1:1) Rf: 0.57

C₂₈H₅₃NO₁₀ MW: 563.72

¹H-NMR (500 MHz, Me₂SO-d₆, 60° C.)

δ=5.460 (br.s, 1H, H-5a), 4.794-4.756 (m, 3H, OCH₂), 4.728, 4.688 (2d,2H, J=6.6 Hz, 6.1 Hz, OCH₂), 4,601-4.565(m, 3H, OCH₂), 4.155 (br.d, 1H),4.056-3.981 (m, 2H), 3.687 (d, 1H, J=7.8 Hz, 9.5 Hz), 3.343, 3.336,3.269, 3.251 (4s, 12H, 4OCH₃), 2.985 (m, 1H, NCH₂), 1.53-1.20 (m, 12H,6CH₂), 1.251 (5, 9H, CC(CH₃)₃), 0.857 (t, 3H, J=7.1 Hz, CH₃)

(40) Synthesis of Compound 30-2

Hydrochloric acid (4 N, 6 ml) was added to a THF solution (4 ml) ofCompound 29-2 (121 mg, 0.215 mmol), followed by stirring at roomtemperature for 1 day. The solvent of the reaction solution wasevaporated under reduced pressure, and the resulting residue wasazeotroped with ethanol 3 times. The resulting residue was purified bySephadex LH-20 (trade name, manufactured by Pharmacia Biotech)(chloroform:methanol=1:1). Then, the mixture was dissolved in water (0.5ml), and 25% aqueous ammonia (0.5 ml) was added thereto. The reactionsolution was directly purified by silica gel column chromatography(chloroform:methanol:water=60:35:8) to obtain Compound 30-2 (31 mg,50.20%).

Developing solvent of Compound 30-2 (chloroform:methanol:water=60:35:8)Rf: 0.25

C₁₅H₂₉NO₄ MW: 287.40

¹H-NMR (500 M CD₃OD)

δ=5.639 (br.s, 1H, H-5a), 4.178-4.084 (m, 3H), 3.478 (dd, 1H, J=7.6 Hz,10.0 Hz), 3.407 (dd, 1H, J=8.5 Hz, 10.0 Hz), 3.206 (br.d, 1H), 2.742(br.ddd, 1H, NCH₂), 2.555 (br.ddd, 1H, NCH₂), 1.55-1.27 (m, 12H, 6CH₂),0.900 (t, 3H, J=7.1 Hz, CH₃)

(41) Synthesis of Compound 294

In argon atmosphere, a DMF solution (2 ml) of Compound 28 (116.0 mg,0.257 mmol) was ice-cooled, and sodium hydride (60% in oil, 37 mg, 0.925mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, lauryl bromide (92.5 μl, 0.385 mmol) was addeddropwise thereto, followed by stirring for 4 hours while graduallyraising the temperature of the reaction solution to room temperature.The reaction solution was ice-cooled, and methanol (1 ml) was addeddropwise thereto, followed by stirring for 30 minutes. The reactionsolution was diluted with diethyl ether, and saturated sodiumbicarbonate water was added thereto, followed by stirring. Then, theorganic layer was washed with saturated sodium bicarbonate water andsaturated brine in this order and dried over magnesium sulfate, and thesolvent was evaporated under reduced pressure. The resulting residue waspurified by silica gel column chromatography (toluene:ethylacetate=10:1, 0.2% triethylamine) to obtain Compound 294 (149.8 mg,94.1%).

Developing solvent of Compound 29-4 (toluene:ethyl acetate=1:1) Rf: 0.66

C₃₂H₆₁NO₁₀ MW: 619.83

(42) Synthesis of Compound 30-4

Hydrochloric acid (4 N, 8 ml) was added to a THF solution (5 ml) ofCompound 29-4 (149.8 mg, 0.240 mmol), followed by stirring at roomtemperature for 1 day. The solvent of the reaction solution wasevaporated under reduced pressure, and the resulting residue wasazeotroped with ethanol 3 times. The resulting residue was purified bysilica gel column chromatography (chloroform:methanol:water=60:35:8).Then, the mixture was dissolved in water (0.1 ml)-MeOH (0.5 ml), and 25%aqueous ammonia (0.5 ml) was added thereto. Ten minutes thereafter, theprecipitated crystals were collected by filtration to obtain Compound30-4 (72 mg, 87.3%).

Developing solvent of Compound 30-4 (chloroform:methanol water 60:35:8)Rf: 0.37

C₁₉H₃₇NO₄ MW: 343.5

¹H-NMR (500 MHz, CDCl₃: CD₃OD=2:1)

δ=5.612 (br.s, 1H, H-5a), 4.193, 4.103 (br.2d, 2H, H-6a, H-6b), 4.167(m, 1H), 3.551 (dd, 1H, J 7.8 Hz, 10.0 Hz), 3.417 (dd, 1H, J=8.6 Hz,10.0 Hz), 3.214 (br.d, 1H), 2.745 (br.ddd, 1H, NCH₂), 2.539 (br.ddd, 1H,NCH₂), 1.53-1.27 (m, 20H, 10CH₂), 0.886 (t, 3H, J=7.1 Hz, CH₃)

(43) Synthesis of Compound 29-5

In argon atmosphere, a DMF solution (2 ml) of Compound 28 (134.7 mg,0.298 mmol) was ice-cooled, and sodium hydride (60% in oil, 42.9 mg,1.073 mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, myristyl bromide (133.1 μl, 0.447 mmol) was addeddropwise thereto, followed by stirring for 6 hours while graduallyraising the temperature of the reaction solution to room temperature.The reaction solution was ice-cooled, and methanol (1 ml) was addeddropwise thereto, followed by stirring for 30 minutes. The reactionsolution was diluted with diethyl ether, and saturated sodiumbicarbonate water was added thereto, followed by stirring. Then, theorganic layer was washed with saturated sodium bicarbonate water andsaturated brine in this order and dried over magnesium sulfate, and thesolvent was evaporated under reduced pressure. The resulting residue waspurified by silica gel column chromatography (toluene:ethylacetate=10:1, 0.2% triethylamine) to obtain Compound 29-5 (172.9 mg,89.5%).

Developing solvent of Compound 29-5 (toluene:ethyl acetate=1:1) Rf: 0.59

C₃₄H₆₅NO₁₀ MW: 647.88

¹H-NMR (500 MHz, CDCl₃)

δ=5.566 (br.s, 1H, H-5a), 4.921 (br.d, 1H, OCH₂), 4.882-4.807 (m, 3H,OCH₂), 4.743 (d, 1H, J=6.3 Hz, OCH₂), 4.691-4.619 (m, 3H, OCH₂), 3.779(m, 1H), 3,443, 3.433, 3.371, 3.356 (4s, 12H, 4OCH₃), 3.064 (m, 1H,NCH₂), 1.58-1.25 (m, 24H, 12CH₂), 1.256 (s, 9H, CC(CH₃)₃), 0.881 (t, 3H,J=7.1 Hz, CH₃)

(44) Synthesis of Compound 30-5

Hydrochloric acid (4 N, 8.5 ml) was added to a THF solution (5 ml) ofCompound 29-5 (172.9 mg, 0.267 mmol), followed by stirring at roomtemperature for 1 day. The solvent of the reaction solution wasevaporated under reduced pressure, and the resulting residue wasazeotroped with ethanol 3 times. The resulting residue was purified bysilica gel column chromatography (chloroform:methanol:water=60:35:8).Then, the mixture was dissolved in water (0.2 ml), and 25% aqueousammonia (0.2 ml) was added thereto. Ten minutes thereafter, theprecipitated crystals were collected by filtration to obtain Compound30-5 (32.2 mg, 32.5%).

Developing solvent of Compound 30-5 (chloroform:methanol:water=60:35:88)

Rf: 0.38

C₂₁H₄₁NO₄ MW: 371.55

¹H-NMR (500 MHz, CDCl₃: CD₃OD=2:1)

δ=5.609 (br.s, 1H, H-5a), 4.192, 4.103 (br.2d, 2H, H-6a, H-6b), 4.167(m, 1H), 3.551 (dd, 1H, J=7.6 Hz, 10.0 Hz), 3.424 (t, 1H, J=9.2 Hz),3.225 (br.d, 1H), 2.753 (br.ddd, 1H, NCH₂), 2.550 (br.ddd, 1H, NCH₂),1.53-1.26 (m, 24H, 12CH₂), 0.885 (t, 3H, J=7.1 Hz, CH₃)

(45) Synthesis of Compound 29-6

In argon atmosphere, a Do solution (1.5 ml) of Compound 28 (100.1 mg,0.222 mmol) was ice-cooled, and sodium hydride (60% in oil, 32 mg, 0.798mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, 1-bromooctadecane (110.9 mg, 0.333 mmol) was addedthereto, followed by stirring for 4 hours while gradually raising thetemperature of the reaction solution to room temperature. The reactionsolution was ice-cooled, and methanol (1 ml) was added dropwise thereto,followed by stirring for 30 minutes. The reaction solution was dilutedwith diethyl ether, and saturated sodium bicarbonate water was addedthereto, followed by stirring. The organic layer was washed withsaturated sodium bicarbonate water and saturated brine in this order anddried over magnesium sulfate, and the solvent was evaporated underreduced pressure. The resulting residue was purified by silica gelcolumn chromatography (toluene:ethyl acetate=8:1, 0.2% triethylamine) toobtain Compound 29-6 (111.6 mg, 71.5%).

Developing solvent of Compound 29-6 (toluene:ethyl acetate=1:1) Rf: 0.60

C₃₈H₇₃NO₁₀ MW: 703.99

¹H-NMR (500 MHz, CDCl₃)

δ=5.566 (br.s, 1H, H-5a), 4.923 (br.d, 1H, OCH₂), 4.883-4.806 (m, 3H,OCH₂), 4.743 (d, 1H, J=6.6 Hz, OCH₂), 4.658-4.618 (m, 3H, OCH₂), 3.762(m, 1H), 3.443, 3.356 (2s, 12H, 4OCH₃), 3.064 (m, 1H, NCH₂), 1.61-1.26(m, 32H, 16CH₂), 1.255 (s, 9H, CC(CH₃)₃), 0.880 (t, 3H, J=7.1 Hz, CH₃)

(46) Synthesis of Compound 30-6

Hydrochloric acid (4 N, 4 ml) was added to a THF solution (2.5 ml) ofCompound 29-6 (81.9 mg, 0.116 mmol), followed by stirring at roomtemperature for 1 day, and further stirring at 35° C. for 2 hours. Thesolvent of the reaction solution was evaporated under reduced pressure,and the resulting residue was azeotroped with ethanol 3 times. Theresulting residue was purified by silica gel column chromatography(chloroform:methanol:water=60:35:8). Then, the mixture was dissolved inmethanol (0.5 ml)-water (0.5 ml), and 25% aqueous ammonia (0.5 ml) wasadded thereto. Ten minutes thereafter, the thus precipitated crystalswere collected by filtration to obtain Compound 30-6 (54 mg, 79.8%).

Developing solvent of Compound 30-6 (chloroform:methanol:water=60:35:8)Rf: 0.30

C₂₅H₄₉NO₄ MW: 427.66

¹H-NMR (500 MHz, CDCl₃: CD₃OD=2:1)

δ=5.609 (br.s, 1H, H-5a), 4.192, 4.101 (br.2d, 2H, H-6a, H-6b), 4.170(m, 1H), 3.553 (dd, 1H, J=7.6 Hz, 10.0 Hz), 3.415 (dd, 1H, J=8.5 Hz,10.0 Hz), 3.211 (br.d, 1H), 2.743 (br.ddd, 1H, NCH₂), 2.536 (br.ddd, 1H,NCH₂), 1.53-1.23 (m, 32H, 16CH₂), 0.885 (t, 3H, J=7.0 Hz, CH₃)

(47) Synthesis of Compound 29-7

In argon atmosphere, a DMF solution (1.5 ml) of Compound 28 (80 mg,0.177 mmol) was ice-cooled, and sodium hydride (60% in oil, 25.5 mg,0.638 mmol) was added thereto, followed by stirring for 10 minutes underice-cooling. Then, bromodocosane (103.5 mg, 0.266 mmol) was addedthereto, followed by stirring for 2 hours while gradually raising thetemperature of the reaction solution to room temperature. The reactionsolution was ice-cooled, and methanol (1 ml) was added dropwise thereto,followed by stirring for 30 minutes. The reaction solution was dilutedwith diethyl ether, and saturated sodium bicarbonate water was addedthereto, followed by stirring. Then, the organic layer was washed withsaturated sodium bicarbonate water and saturated brine in this order anddried over magnesium sulfate, and the solvent was evaporated underreduced pressure. The resulting residue was purified by silica gelcolumn chromatography (toluene:ethyl acetate=10:1, 0.2% triethylamine)to obtain Compound 29-7 (39.5 mg, 29.4%).

Developing solvent of Compound 29-7 (toluene:ethyl acetate=1:1) Rf: 0.56

C₄₂H₈₁NO₁₀ MW: 760.10

(48) Synthesis of Compound 30-7

Hydrochloric acid (4 N, 1.5 ml) was added to a THF solution (1.0 ml) ofCompound 29-7 (39.5 mg, 0.052 mmol), followed by stirring at 40° C. for1 day. The solvent of the reaction solution was evaporated under reducedpressure, and the resulting residue was azeotroped with ethanol 3 times.The resulting residue was purified by silica gel column chromatography(chloroform:methanol:water=60:35:8). Then, the mixture was dissolved inmethanol (0.5 ml)-water (0.5 ml), and 25% aqueous ammonia (0.5 ml) wasadded thereto. Ten minutes thereafter, the thus precipitated crystalswere collected by filtration to obtain Compound 30-7 (25 mg, 99%)

Developing solvent of Compound 30-7 (chloroform:methanol:water=60:35:8)Rf: 0.30

C₂₉H₅₇NO₄ MW: 483.77

¹H-NMR (500 MHz, CDCl₃: CD₃OD=2:1)

δ=5.609 (br.s, 1H, H-5a), 4.192, 4.102 (br.2d, 2H, H-6a, H-6b), 4.167(m, 1H), 3.553 (dd, 1H, J=7.8 Hz, 10.0 Hz), 3.416 (dd, 1H, J=8.3 Hz,10.0 Hz), 3.212 (br.d, 1H), 2.744 (br.ddd, 1H, NCH₂), 2.537 (br.ddd, 1H,NCH₂), 1.53-1.26 (m, 40H, 20CH₂), 0.886 (t, 3H, J=7.1 Hz, CH₃)

<2> Synthesis of Substances of the Invention

(1) Synthesis of Substance A-1 of the Invention

Compound 25-1 (16.5 mg, 0.0713 mmol) was suspended in 3 ml of water, and0.5 ml of concentrated hydrochloric acid was added thereto, followed bystirring for 1 hour. Then, the mixture was evaporated for drying underreduced pressure at 50° C., and ethanol and toluene were added theretoto carry out azeotropy 3 times. The residue was dissolved in water andfreeze-dried to obtain Substance A-1 of the invention (14.7 mg, 77%) asa colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.731 (br.d, 1H, H-5a), 4.259 (br.d, 1H, H-6a), 4.165 (d, 1H, J=4.0Hz, H-1), 4.146 (br.d, 1H, H-6b), 3.947 (dd, 1H, J=8.1, 9.8 Hz, H-3),3.691 (br.d, 1H, H-4), 3.543 (dd, 1H J=4.21, 9.8 Hz, H-2), 3.094(br.ddd, 2H, H-1′), 1.74-1.28 (m, 4H, 2CH₂), 1.008 (t, 3H, J=7.3 Hz,CH₃)

(2) Synthesis of Substance A-2 of the Invention

Compound 25-2 (51.3 mg) was suspended in 12 ml of water, and 0.2 ml ofconcentrated hydrochloric acid was added thereto, followed by stirringfor 5 minutes. Then, the mixture was evaporated at 50° C., and ethanoland toluene were added thereto to carry out azeotropy 3 times. Theresidue was dissolved in water and freeze-dried to obtain Substance A-2of the invention (51.7 mg) as a colorless solid.

Melting point; 80.9-82.3° C.

[α]²⁰ _(D)=+9.83° (c=1.0 methanol)

¹H-NMR (500 MHz, 1:2=CD₃OD: CDCl₂)

δ=5.734 (br.d, 1H, H-5a), 4.268 (brd, 1H, H-6a), 4.153 (d, 1H, J=4.1 Hz,H-1), 4.133 (br.d, 1H, H-6b), 3.953 (dd, 1H, J=8.1, 9.8 Hz, H-3), 3.638(br.d, 1H, H-4), 3.549 (dd, 1H, J=4.1, 9.8 Hz, H-2), 3.063 (m, 2H,H-1′×2), 1.75, 1.26 (m, 12H, 6CH₂, H-2′-7′), 0.894 (t, 3H, J=7.1 Hz,CH₃, H-8′)

(3) Synthesis of Substance A-3 of the Invention

Compound 25-3 (15.0 mg, 0.0476 mmol) was suspended in 10 ml of water,and 0.5 ml of concentrated hydrochloric acid was added thereto, followedby stirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceA-3 of the invention (16.7 mg, 99%) as a colorless solid.

¹H-NMR (500 M CD₃OD)

δ=5.737 (br.d, 1H, H-5a), 4.255 (br.d, 1H, H-6a), 4.165 (d, 1H, J=4.2Hz, H-1), 4.145 (br.d, 1H, H-6b), 3.950 (dd, 1H, J=8.3, 9.8 Hz, H-3),3.696 (br.d, 1H, H-4), 3.543 (dd, 1H, J=4.0, 9.8 Hz, H-2), 3.088(br.ddd, 2H, H-1′), 1.76-1.29 (m, 16H, 8CH₂), 0.894 (t, 3H, J=7.1 Hz,CH₃)

(4) Synthesis of Substance A-4 of the Invention

Compound 25-4 (15.0 mg, 0.0437 mmol) was suspended in 10 ml of water,and 0.5 ml of concentrated hydrochloric acid was added thereto, followedby stirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceA-4 of the invention (16.6 mg, 100%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.730 (br.d, 1H, H-5a), 4.255 (br.d, 1H, H-6a), 4.163 (d, 1H, J=3.9Hz, H-1), 4.144 (br.d, 1H, H-6b), 3.942 (dd, 1H, J=8.3, 9.8 Hz, H-3),3.690 (br.d, 1H, H-4), 3.540 (dd, 1H, J=3.9, 9.8 Hz, H-2), 3.086(br.ddd, 2H, H-1′), 1.75-1.29 (m, 20H, 10CH₂), 0.893 (t, 3H, J=7.1 Hz,CH₃)

(5) Synthesis of Substance A-5 of the Invention

Compound 25-5 (15.0 mg, 0.0404 mmol) was suspended in 10 ml of water,and 0.5 ml of concentrated hydrochloric acid was added thereto, followedby stirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceA-5 of the invention (7.2 mg, 44%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.723 (br.d, 1H, H-5a), 4.252 (br.d, 1H, H-6a), 4.159 (d, 1H, J=4.7Hz, H-1), 4.141 (br.d, 1H, H-6b), 3.933 (dd, 1H, J=8.1, 9.8 Hz, H-3),3.683 (br.d, 1H, H-4), 3.532 (dd, 1H, J=4.0, 9.8 Hz, H-2), 3.082(br.ddd, 2H, H-1′), 1.74-1.28 (m, 24H 12CH₂), 0.893 (t, 3H, J=7.1 Hz,CH₃)

(6) Synthesis of Substance A-6 of the Invention

Compound 25-6 (13.2 mg, 0.0309 mmol) was suspended in 10 ml of water,and 0.5 ml of concentrated hydrochloric acid was added thereto, followedby stirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceA-6 of the invention (14.3 mg, 99%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.708 (br.d, 1H, H-5a), 4.259 (br d, 1H, H-6a), 4.159 (d, 1H, J=4.2Hz, H-1), 4.142 (br.d, 1H, H-6b), 3.925 (dd, 1H, J=8.3, 9.8 Hz, H-3),3.670 (br d, 1H, H-4), 3.533 (dd, 1H, J=3.9, 9.8 Hz, H-2), 3.080(br.ddd, 2H, H-1′), 1.74-1.28 (m, 32H, 16CH₂), 0.892 (t, 3H, J=7.1 Hz,CH₃)

(7) Synthesis of Substance A-7 of the Invention

Compound 25-7 (13.5 mg, 0.0279 mmol) was suspended in 10 ml of water,and 0.5 ml of concentrated hydrochloric acid was added thereto, followedby stirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceA-7 of the invention (14.5 mg, 100%) as a colorless solid.

¹H-NM (500 MHz, CD₃OD)

δ=5.710 (br.d, 1H, H-5a), 4.255 (br.d, 1H, H-6a), 4.157 (d, 1H, J=4.2Hz, H-1), 4.140 (br.d, 1H, H-6b), 3.924 (dd, 1H, J=8.3, 9.8 Hz, H-3),3.675 (br.d, 1H, H-4), 3.529 (dd, 1H, J=4.2, 9.8 Hz, H-2), 3.081(br.ddd, 2H, H-1′), 1.74-1.25 (m, 40H, 20CH₂), 0.893 (t, 3 H, J=7.1 Hz,CH₃)

(8) Synthesis of Substance B-1 of the Invention

Compound 30-1 (15 mg, 0.0649 mmol) was suspended in 10 ml of water, and0.5 ml of concentrated hydrochloric acid was added thereto, followed bystirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceB-1 of the invention (16 mg, 92%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.672 (br.s, 1H, H-5a), 4.250, 4.190 (2br.d, 2H, H-6a, H-6b), 4.124(m, 1H, H-1), 3.814 (m, 1H, H-4), 3.691 (dd, 1H, J=8.8, 9.9 Hz, H-3),3.533 (dd, 1H, J=8.1, 9.8 Hz, H-2), 3.106 (t, 2H, J=8.1 Hz, H-1′a,H-1′b), 1.75-1.27 (m, 4H, 2CH₂), 1.005 (t, 3H, J=7.3 Hz, CH₃)

(9) Synthesis of Substance B-2 of the Invention

Compound 30-2 (22.4 mg, 0.0780 mmol) was suspended in 8 ml of water andmixed with 1.0 ml of concentrated hydrochloric acid, followed bystirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceB-2 of the invention (18.3 mg, 72%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.659 (br.s, 1H, H-5a), 4.250, 4.138 (2br.d, 2H, H-6a, H-6b), 4.105(m, 1H, H-1), 3.802 (m, 1H, H-4), 3.656 (dd, 1H, J=8.8, 9.8 Hz, H-3),3.515 (dd, 1H, J=7.9, 9.9 Hz, H-2), 3.089 (t, 2H, J=8.1 Hz, H-1′a,H-1′b), 1.73-1.31 (m, 12H, 6CH₂), 0.905 (t, 3H, J=7.0 Hz, CH₃)

(10) Synthesis of Substance B-4 of the Invention

Compound 30-4 (15 mg, 0.0437 mmol) was suspended in 10 ml of water, and0.5 ml of concentrated hydrochloric acid was added thereto, followed bystirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceB-4 of the invention (I 5 mg, 90%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.658 (br.s, 1H, H-5a), 4.251, 4.188 (2br.d, 2H, H-6a, H-6b), 4.117(m, 1H, H-1), 3.800 (m, 1H, H-4), 3.676 (dd, 1H, J=8.8, 9.8 Hz, H-3),3.528 (dd, 1H, J=7.8, 9.8 Hz, H-2), 3.090 (t, 2H, J=8.1 Hz, H-1′a,H-1′b), 1.76-1.28 (m, 20H, 10CH₂), 0.892 (t, 3H, J=7.1 Hz, CH₃)

(11) Synthesis of Substance B-5 of the Invention

Compound 30-5 (15 mg, 0.0404 mmol) was suspended in 10 ml of water, and0.5 ml of concentrated hydrochloric acid was added thereto, followed bystirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceB-5 of the invention (16 mg, 97%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.652 (brs, 1H, H-5a), 4.253, 4.192 (2br.d, 2H, H-6a, H-6b), 4.121 (m,1H, H-1), 3.794 (m, 1H, H-4), 3.679 (t, 1H, J=9.8 Hz, H-3), 3.533 (dd,1H, J=8.1, 9.8 Hz, H-2), 3.087 (t, 2H, J=8.1 Hz, H-1′a, H-1′b),1.74-1.27 (m, 24H, 12CH₂), 0.891 (t, 3H, J=7.1 Hz, CH₃)

(12) Synthesis of Substance 13-6 of the Invention

Compound 30-6 (15 mg, 0.0351 mmol) was suspended in 10 ml of water, and0.5 ml of concentrated hydrochloric acid was added thereto, followed bystirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceB-6 of the invention (14 mg, 86%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.647 (br.s, 1H, H-5a), 4.254, 4.186 (2br.d, 2H, H-6a, H-6b), 4.110(m, 1H, H-1), 3.793 (m, 1H, H-4), 3.661 (t, 1H, J=9.8 Hz, H-3), 3.524(dd, 1H, J=7.8, 9.8 Hz, H-2), 3.085 (t, 2H, J=8.1 Hz, H-1′a, H-1′b),1.74-1.27 (m, 32H, 16CH₂), 0.892 (t, 3H, J=7.1 Hz, CH₃)

(13) Synthesis of Substance B-7 of the Invention

Compound 30-7 (15 mg, 0.0310 mmol) was suspended in 10 ml of water, and0.5 ml of concentrated hydrochloric acid was added thereto, followed bystirring for 1 hour. Then, the mixture was evaporated at 50° C., andethanol and toluene were added thereto to carry out azeotropy 3 times.The residue was dissolved in water and freeze-dried to obtain SubstanceB-7 of the invention (16 mg, 99%) as a colorless solid.

¹H-NMR (500 MHz, CD₃OD)

δ=5.643 (br.s, 1H, H-5a), 4.257, 4.185 (2br.d, 2H, H-6a, H-6b), 4.108(m, 1H, H-1), 3.792 (m, 1H, H-4), 3.651 (t, 1H, J=9.8 Hz, H-3), 3.521(dd, 1H, J=7.8, 10.0 Hz, H-2), 3.084 (t, 2H, J=8.1 Hz, H-1′a, H-1′b),1.73-1.27 (m, 40H, 20CH₂), 0.893 (t, 3H, J=7.1 Hz, CH₃)

<3> Measurement of Neutral β-galactosidase Inhibitory Activity

Each of Substances A-2 to 7 and B-2 to 7 of the invention dissolved inwater for injection, to which DMSO had been added and dissolved therein,if necessary, was diluted with 0.02 M HEPES buffer (pH 7.3) to give aconcentration of 0.05, 0.125, 0.25, 0.5, 1.0, 2.5 or 5.0 μM and added at25 μl into a 96 well microplate. Then, a 0.02 M HEPES buffer (pH 7.3)solution of bovine liver derived β-D-galactosidase (manufactured bySigma) was added at 25 μl, 4-methylumbelliferyl-β-D-galactopyranoside(10 μM) was further added as a fluorescence substrate at 50 μl, followedby incubation at 37° C. for 30 minutes. Then, the reaction was stoppedby adding 100 μl of 2 M Na₂CO₃. An amount of 4-methylumbelliferonereleased by the enzyme reaction (fluorescence intensity) was measured(excitation wavelength 355 nm, measuring wavelength 460 nm) by afluorescence reader (product name: ARVOSX manufactured by WALLAC). Anamount of the 4-methylumbelliferone released at the time of not addingthe inhibitor was defined as 100%, and quantitative change in the4-methylumbelliferone formed by addition of each substance to beevaluated was relatively evaluated. Results of Substances A-2 to 7 ofthe invention are shown in FIG. 2, and results of Substances B-2 to 7 ofthe invention in FIG. 3. In addition, as the 50% inhibitionconcentration (IC₅₀ value), concentration of each substance of theinvention which reduces 50% of the concentration of4-methylumbelliferone at the time of un-adding each substance of theinvention was calculated from an inhibition curve.

In this connection, regarding those which have high inhibitory activityamong the substances of the invention, their IC₅₀ values were calculatedby carrying out the enzyme inhibition measurement by further dilutingthem to a concentration of 0.0025, 0.005, 0.01 or 0.025 μM. The resultsare shown in Table 3. TABLE 3 50% Inhibition concentration Substances ofthe invention (IC₅₀ value, μM) Substance A-2 0.377    Substance A-30.0499  Substance A-4 0.014 (*) Substance A-5 0.005 (*) Substance A-60.025 (*) Substance A-7 1.402    Substance B-2 0.096 (*) Substance B-4<0.005 (*)  Substance B-5 0.006 (*) Substance B-6 0.017 (*) SubstanceB-7 0.067   (*) Measured to 0.0025, 0.005, 0.01 and 0.025 μM<4> Measurement of Acidic β-galactosidase Inhibitory Activity

Each of Substances A-2 to 7 of the invention dissolved in water forinjection, to which DMSO had been added and dissolved therein, ifnecessary, was diluted with a 0.2 M acetate buffer (pH 4.4) to give aconcentration of 0.05, 0.125, 0.25, 0.5, 1.0, 2.5 or 5.0 μM and added at25 μl into a 96 well microplate. Then, a 0.2 M acetate buffer (pH 4.4)solution of bovine sperm-derived β-D-galactosidase (manufactured bySigna) was added at 25 μl, and4-methylumbelliferyl-β-D-galactopyranoside (10 μM) was further added asa fluorescence substrate at 50 μl, followed by incubation at 25° C. for30 minutes. Then, the reaction was stopped by adding 100 μl of 2 MNa₂CO₃, and an amount of 4-methylumbelliferone released by the enzymereaction (fluorescence intensity) was measured (excitation wavelength355 nm, measuring wavelength 460 nm) by a fluorescence reader (productname; ARVOSX manufactured by WALLAC). An amount of the4-methylumbelliferone released at the time of not adding the inhibitorwas defined as 100%, and quantitative change in the4-methylumbelliferone formed by addition of each substance to beevaluated was relatively evaluated. The results are shown in FIG. 4. Inaddition, as the 50% inhibition concentration (IC₅₀ value), theconcentration of each substance of the invention which reduces 50% ofthe concentration of 4-methylumbelliferone at the time of un-adding eachsubstance of the invention was calculated from an inhibition curve. Theresults are shown in Table 4. TABLE 4 50% Inhibition concentrationSubstances of the invention (IC₅₀ value, μM) Substance A-2 of theinvention 0.476 Substance A-3 of the invention 0.301 Substance A-4 ofthe invention 1.432 Substance A-5 of the invention 2.689 Substance A-6of the invention 1.791 Substance A-7 of the invention —<5> Measurement of Neutral β-glucosidase Inhibitory Activity

Each of Substances A-2 to 7 and B-2 to 7 of the invention dissolved inwater for injection, to which DMSO had been added and dissolved therein,as occasion demands, was diluted with 0.02 M HEPES buffer (pH 7.3) togive a concentration of 0.0025, 0.005, 0.01, 0.025, 0.05, 0.125, 0.25,0.5, 1.0, 2.5 or 5.0 μM and added at 25 μl into a 96 well microplate.Then, a 0.02 M HEPES buffer (pH 7.3) solution of bovine liver-derivedβ-D-galactosidase (manufactured by Sigma) was added at 25 μl, and4-methylumbelliferyl-β-D-galactopyranoside (10 μM) was further added asa fluorescence substrate at 50 μl, followed by incubation at 37° C. for30 minutes. Then, the reaction was stopped by adding 100 μl of 2 MNa₂CO₃, and an amount of 4-methylumbelliferone released by the enzymereaction (fluorescence intensity) was measured (excitation wavelength355 nm, measuring wavelength 460 nm) by a fluorescence reader (productname: ARVOSX, manufactured by WALLAC). An amount of the4-methylumbelliferone released at the time of not adding the inhibitorwas defined as 100%, and quantitative change in the4-methylumbelliferone formed by addition of each substance to beevaluated was relatively evaluated. Results of Substances A-2 to 7 ofthe invention are shown in FIG. 5, and results of Substances B-2 to 7 ofthe invention in FIG. 6. In addition, as the 50% inhibitionconcentration (IC₅₀ value), the concentration of each substance of theinvention which reduces 50% of the concentration of4-methylumbelliferone at the time of un-adding each substance of theinvention was calculated from an inhibition curve. The results are shownin Table 5. TABLE 5 50% Inhibition concentration Substances of theinvention (IC₅₀ value, μM) Substance A-2 0.028 Substance A-3 0.007Substance A-4 0.006 Substance A-5 0.014 Substance A-6 0.013 SubstanceA-7 0.059 Substance B-2 0.152 Substance B-4 0.015 Substance B-5 <0.003Substance B-6 0.033 Substance B-7 1.043<6> Solubility Test

When solubility of Substance A-2 of the invention was confirmed, itdissolved in distilled water at a concentration of 300 mg/ml and 24° C.On the other hand, Compound 25-2 was not dissolved in water at aconcentration of 2 mg/ml and 24° C. and dissolved at 40° C., butCompound 25-2 precipitated when returned again to 24° C.

In addition, the solubility of Compound 30-2 and the substance B-2 ofthe invention was confirmed by adding 50 μl of distilled water to 5.4 mgof each sample at 19° C. Substance B-2 of the invention was completelydissolved when 50 μl of distilled water was added (>108 mg/ml). On theother hand, Compound 30-2 was not dissolved by addition of 50 μl ofdistilled water, and completely dissolved when 1.95 ml of the same wasadded (2.8 mg/ml).

While the invention has been describe in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

This application is based on a Japanese patent application filed on May19, 2003 (Japanese Patent Application No. 2003-140868), entire contentsthereof being thereby incorporated by reference.

INDUSTRIAL APPLICABILITY

Acid addition salts of novel carba-sugar amine derivatives havinginhibitory activity upon β-galactosidase or β-glucosidase and withimproved solubility are provided by the invention. In addition, theabove-described acid addition salts of novel carba-sugar aminederivatives can be used for the excellent treatment or prevention ofglycolipid metabolic disorders based on the β-galactosidase orβ-glucosidase gene.

1. An acid addition salt of a carba-sugar amine derivative representedby the following formula (1):

wherein R¹ and R² each independently represents a hydrogen atom, or analkyl group, an alkenyl group, an alkynyl group, an acyl group, an arylgroup or an aralkyl group which may have one or at least two of thefollowing substituent (I) or (II), or R¹ and R² are taken together torepresent a substituent (III);

wherein R⁸ to R¹² each independently represents an alkyl group, analkenyl group, an alkynyl group, an acyl group, an aryl group or anaralkyl group; with the proviso that both R¹ and R² are not a hydrogenatom at the same time; R³, R⁴ and R⁷ each independently represents ahydroxyl group or a hydroxyl group having a substituent; and R⁵ and R⁶each independently represents a hydrogen atom, a hydroxyl group or ahydroxyl group having a substituent, with the proviso that when one ofR⁵ and R⁶ is a hydrogen atom, the other is a hydroxyl group or ahydroxyl group having a substituent.
 2. An acid addition salt of acarba-sugar amine derivative represented by the following formula(1)-A-2:

wherein R¹ and R² each represents as defined in claim
 1. 3. An acidaddition salt of a carba-sugar amine derivative represented by thefollowing formula (1)-B-2:

wherein R¹ and R² each represents as defined in claim
 1. 4. The acidaddition salt of a carba-sugar amine derivative according to any one ofclaims 1 to 3, which is hydrochloride or sulfate.
 5. A medicament whichcomprises the acid addition salt of a carba-sugar amine derivativeaccording to any one of claims 1 to 4 as an active ingredient.
 6. Themedicament according to claim 5, which is a preventive agent or atherapeutic agent for glycolipid metabolic disorders.
 7. A process forproducing an acid addition salt of a carba-sugar amine derivative, whichcomprises allowing a carba-sugar amine derivative represented by thefollowing formula (1) to contact with an acid in an aqueous solvent toobtain the acid addition salt thereof:

wherein R¹ and R² each independently represents a hydrogen atom, or analkyl group, an alkenyl group, an alkynyl group, an acyl group, an arylgroup or an aralkyl group which may have one or at least two of thefollowing substituent (I) or (II), or R¹ and R² are taken together torepresent a substituent (III):

wherein R⁸ to R¹² each independently represents an alkyl group, analkenyl group, an alkynyl group, an acyl group, an aryl group or anaralkyl group; with the proviso that both R¹ and R² are not a hydrogenatom at the same time; R³, R⁴ and R⁷ each independently represents ahydroxyl group or a hydroxyl group having a substituent; and R⁵ and R⁶each independently represents a hydrogen atom, a hydroxyl group or ahydroxyl group having a substituent, with the proviso that when one ofR⁵ and R⁶ is a hydrogen atom, the other is a hydroxyl group or ahydroxyl group having a substituent.